Dutpase inhibitors

ABSTRACT

Deoxyuridine derivatives of the formula 
     
       
         
         
             
             
         
       
         
         
           
             where 
             A is O, S or CH 2 ; B is O, S or CHR 3 ; 
             R 1  is H, or various substituents; 
             R 2  is H, F; 
             R 3  is H, F, OH, NH 2 ; or R 2  and R 3  together form a chemical bond; 
             D is —NHCO—, —CONH—, —O—, —C(═O)—, —CH═CH, —C ═ C—, —NR 5 —; R 4  is hydrogen or various substituents; 
             R 5  is H, C 1 -C 4  alkyl, C 1 -C 4  alkanoyl; 
             E is Si or C; 
             R 6 , R 7  and R 8  are independently selected from C 1 -C 8  alkyl, C 2 -C 8  alkenyl, C 2 -C 8  alkynyl, or a stable monocyclic, bicyclic or tricyclic ring system
 
have utility in the prophylaxis o treatment of parasitic diseases such as malaria

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of co-pending application Ser. No.10/585,281, filed on Jul. 3, 2006, which is a national phase under 35U.S.C. §371 of PCT International Application No. PCT/GB/2005/050001which has an international filing date of Jan. 6, 2005, which designatedthe United States and on which priority is claimed under 35 U.S.C. §120,the entire contents of which are hereby incorporated by reference. ThisDivisional application claims priority under 35 U.S.C. §119 onApplication 0400290.3 filed in the United Kingdom on Jan. 8, 2004, theentire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceuticals active against parasitedUTPase and methods for treating parasitical infections, especiallymalaria, by administering such compounds.

TECHNICAL BACKGROUND

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) E.C. 3.6.1.23 isan ubiquitous enzyme which hydrolyzes deoxyuridine triphosphate (dUTP)to deoxyuridine monophosphate (dUMP) and pyrophosphate, typically in thepresence of magnesium ions. This reaction is thought to occur primarilyto limit pools of intracellular dUTP in order to prevent significanturidine incorporation into DNA during replication and repair. A secondrole of dUTPase is to provide substrate (dUMP) for the de novo synthesisof thymidylate.

Two groups of researchers, McIntosh et al., PNAS, 89:8020-8024 (1992)and Strahler et al., PNAS, 90:4991-4995 (1993), have reportedly isolatedthe trimeric human dUTPase enzyme and characterized the enzyme by itscDNA and amino acid sequences.

McIntosh reported a cDNA of 526 base pairs containing an ORF whichencoded a protein of 141 amino acids and a 3 f flanking sequencefollowing the ORF. Strahler reported the identical cDNA and amino acidsequence as did McIntosh, with the exception of two additional bases atthe 51 end of the cDNA and a longer 3 f flanking sequence. The humandUTPase reported by both groups was found to have a high degree ofhomology with dUTPase from other organisms including that from yeasts,bacteria and viruses. Strahler further reported that human dUTPaseexists in both, phosphorylated and a non-phosphorylated forms.

International patent application no WO97/36916 discloses the sequence ofnuclear and mitochondrial isoforms of dUTPase.

In both prokaryotic and eukaryotic cell systems, dUTPase has beenclearly shown to be an essential enzyme, without which the cell willdie. Lack of dUTPase leads to elevated cellular dUTP pools, resulting inan increased misincorporation of uridine into DNA. In addition toprokaryotes and eukaryotes, a number of viruses, such as herpes simplex,are known to encode a dUTPase function.

International patent application no WO95/15332 proposes a range ofuridine di- and triphosphate analogues in which the oxygen atoms betweenphosphate groups are replaced with methylene, secondary amine ortertiary amine, and/or oxo functions on the phosphate are replaced withsulphur. These compounds are postulated as cytostatics for use againstrapidly growing cancer cells and/or antivirals against herpes.Substantially similar compounds are disclosed in Zalud et al Adv. Exp.Med. Biol. 1995 370 135-138 and Persson et al Bioiorg Med Biochem 1996 4553-556. It should be noted, however that these compounds have beenprimarily designed for crystallographic purposes and the analysis ofenzyme kinetics. These compounds therefore do not possessphysicochemical attributes suggestive of a drug.

The present inventors have established that the substrate specificity ofthe dUTPases of certain protozoal and bacterial parasites of man differfrom the corresponding human cellular and mitochondrial enzymes to suchan extent that a specific set of inhibitor compounds can be preparedwhich selectively inhibit the parasite dUTPase without substantiallyinhibiting the human counterparts. Examples of such parasites includePlasmodium species especially P. falciparum responsible for malaria,Mycobacterial species, especially M. tuberculosum responsible fortuberculosis and Leishmania spp.

Hidalgo-Zarco and González-Pacanowska Current Protein and PeptideScience, 2001, 2, 389-397 describe the isolation and characterisation oftrypanosomal dUTPases. In contrast to the trimeric form of dUTPaseshared by human and malarial enzymes, the trypanosomal enzyme is adimmer. Competitive inhibition of Leishmania dUTPase was shown by thetriphosphate substrate analogue α-β-imido-dUTP, whereas no inhibition ofthat parasite was apparent in the case of5′-O-(4-4′-dimethoxytrityl)-2′-deoxyuridine.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with a first aspect of the invention there are provideduse of deoxyuridine derivatives of the formula I, in the manufacture ofa medicament for the treatment or prophylaxis of parasitic infections,particularly plasmodium infections in mammals, including man.

where

A is O, S or CH₂;

B is O, S or CHR³;

R¹ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl or a 5 or 6 membered,saturated or unsaturated ring containing 0 to 3 heteroatoms selectedfrom N, O and S, the alkyl, alkenyl, alkynyl or ring is optionallysubstituted with R⁴;

R² is H or F;

R³ is H, F, OH, NH₂ or a pharmaceutically acceptable ester, amide orether thereof; or

R² and R³ together form a chemical bond;

D is —NHCO—, —CONH—, —O—, —C(═O)—, —CH═CH, —C═C—, —NR⁵—,

R⁴ is independently hydrogen, halo, cyano, amino, nitro, carboxy,carbamoyl, hydroxy, oxo, C₁-C₅ alkyl, C₁-C₅ haloalkyl, C₁-C₅ alkyloxy,C₁-C₅ alkanoyl, C₁-C₅ alkanoyloxy, C₁-C₅ alkylthio, —N(C₀-C₃-alkyl)₂,hydroxymethyl, aminomethyl, carboxymethyl; —SO₂N(C₀-C₃-alkyl),—SO₂C₁-C₅-alkyl, where n is 1 or 2;

R⁵ is H, C₁-C₃-alkyl, C₁-C₃-alkanoyl;

E is Si or C;

R⁶, R⁷ and R⁸ are independently selected from C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, or a stable monocyclic, bicyclic or tricyclicring system which is saturated or unsaturated in which each ring has 0to 3 heteroatoms selected from N, O and S, wherein any the R⁶, R⁷ and/orR⁸ group may be optionally substituted with R⁴;

and pharmaceutically acceptable salts thereof.

According to a second aspect of the invention there are provided novelcompounds of the formula II

where

A is O, S or CH₂;

B is O, S or CHR³;

R¹ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl or a 5 or 6 membered,saturated or unsaturated ring containing 0 to 3 heteroatoms selectedfrom N, O and S, the alkyl, alkenyl, alkynyl or ring is optionallysubstituted with R⁴;

R² is H or F;

R³ is H, F, OH, NH₂ or a pharmaceutically acceptable ester, amide orether thereof; or

R² and R³ together form a chemical bond;

D is —NHCO—, —CONH—, —O—, —C(═O)—, —CH═CH, —C═C—, —NR⁵—,

R⁴ is independently hydrogen, halo, cyano, amino, nitro, carboxy,carbamoyl, hydroxy, oxo, C₁-C₅ alkyl, C₁-C₅ haloalkyl, C₁-C₅ alkyloxy,C₁-C₅ alkanoyl, C₁-C₅ alkanoyloxy, C₁-C₅ alkylthio, —N(C₀-C₃-alkyl)₂,hydroxymethyl, aminomethyl, carboxymethyl; —SO_(n)N(C₀-C₃-alkyl),—SO_(n)C₁-C₅ alkyl, where n is 1 or 2;

R⁵ is H, C₁-C₃ alkyl, C₁-C₃ alkanoyl;

E is Si or C;

R⁶ and R⁷ are independently selected from a stable monocyclic, bicyclicor tricyclic ring system which has an aromatic nature in which each ringhas 0 to 3 heteroatoms selected from N, O and S;

R⁸ selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or a stablemonocyclic, bicyclic or tricyclic ring system which is saturated orunsaturated in which each ring has 0 to 3 heteroatoms selected from N, Oand S; wherein

R⁶, R⁷ and R⁸ group are optionally substituted with R⁴;

with the proviso that if the group C₀-C₃alkyl-D-C₀-C₃ alkyl is —O—CH₂—,then the group E(R⁶)(R⁷)(R⁸) is not trityl, methoxylated trityl ortert.butyldiphenylsilyl;

or a pharmaceutically acceptable salt thereof.

The novel compounds of the invention are useful in methods for thetreatment or propylaxis, or in the manufacture of a medicament for suchtreatment or prophylaxis, of parasitic infections, such as Leishmania,trypansoma, human African trypanosomiasis, Chagas disease or plasmodium(malaria).

The potency and selectivity of the compounds and methods of theinvention, which presuppose substantial lipophilicity at the 5′ positionis surprising bearing in mind that the active site of the dUTPase enzymeis intended to recognize and accommodate highly polar, hydrophilicmoieties, ie the triphosphorylated nucleotides.

Conveniently, A is —O— and B is —CHR³— thus defining a 2′-deoxyriboseanalogues.

Alternative preferred variants include those where A is —O— and B is—O—, or —S—, thus defining a dioxolane or especially an oxathiolanederivative.

Other preferred variants include those wherein R² and R³ form a chemicalbond and A is —O—, thus defining a 2′3′-dideoxy, didehydroribosederivative or R² and R³ are H, thus defining a 2′,3′-dideoxyribosederivative.

Still further preferred variants include those wherein R² and R³ form achemical bond and A is —CH₂—, thus defining a 2-cyclopentene derivativeor those wherein R² and R³ are H, thus defining a cyclopentanederivative.

It is currently preferred that R³ is H, NH₂, OH or F. An alternative,but currently less favoured, R³ is a lipophilic ester such as straightor branched chain alkyl or benzyl ester or an ether such as straight orbranched chain alkyl or benzyl ether or alkylated silyl function.

R¹ is preferably a small substituent, most preferably H.

Favoured C₀-C₃-alkylene-D-C₀-C₃-alkylene configurations includeaminomethylene, aminoethylene and aminopropylene, methylaminomethylene,methylaminoethylene, ethylaminomethylene, —(N-methyl)aminomethylene,—(N-methyl)aminoethylene, —(N-methyl)aminopropylene andmethyl-(N-methyl)aminomethylene. Currently the most preferred is-aminomethylene-. The order of the hetero atom and alkylene moieties inthe indicated groups as used herein corresponds to the configuration ofFIG. I or II as depicted above, that is “aminomethylene” has thenitrogen atom adjacent E and the methylene moiety proximal to the base.

Particularly preferred C₀-C₃-alkylene-D-C₀-C₃-alkylene configurationsinclude —O—, oxymethylene, oxyethylene, oxypropylene methyloxymethyleneand methyloxyethylene. Currently the most preferred in this series is-oxymethylene-.

Preferably at least one of R⁶, R⁷ and/or R⁸ has an aromatic nature,although this tends to be less important if R³ has a lipophilic nature.Conveniently two of R⁶, R⁷ and/or R⁸ have an aromatic nature and theinvention even embraces compounds wherein all three have an aromaticnature.

Ring systems for R⁶, R⁷ and/or R⁸ are typically bonded direct to E, butmay optionally be bonded to E via a methylene linker. For example R⁶ maybe optionally substituted benzyl, thereby representing phenyl bondedthrough a methylene to E.

Ring systems for R⁶, R⁷ and/or R⁸ having an aromatic nature includeoptionally substituted heteroaryls such as furyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, especially pyridyl; and optionallysubstituted carbocycles such as phenyl. Ring systems having an aromaticnature also include multi-ring systems wherein only one ring has anaromatic nature such as indolinyl and ring systems wherein more than onering has an aromatic nature such as naphthyl or any of the aboveheterocyclic rings fused to phenyl, such as benzimidazolyl.

Convenient values for R⁶, R⁷ and/or R⁸ include heterocycles such asfuryl, thienyl, pyranyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl,pyridyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl,thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, especiallypyridyl, and carbocycles such as cycloalkyl, cycloalkenyl and especiallyphenyl. Alternative values for R⁶, R⁷ and/or R⁸ include straight orbranched alkyl, including methyl, ethyl, i-propyl and t-butyl.

The optional substituent(s) to R⁶, R⁷, and/or R⁸ include 1 to 3,preferably 1 substituent per ring, selected from halo, preferablyfluoro, cyano (preferably cyano), amino, nitro, carboxy, carbamoyl,hydroxy, oxo, C₁-C₅ alkyl, preferably methyl or t-butyl, C₁-C₅haloalkyl, preferably trifluoromethyl, C₁-C₅ alkyloxy, preferablymethoxy, C₁-C₅ alkanoyl, preferably acetyl, C₁-C₅ alkanoyloxy,preferably acetoxy, C₁-C₅ alkylthio, —N(C₀-C₃-alkyl)₂, preferably NHMeor NMe, hydroxymethyl, aminomethyl, carboxymethyl; —SO_(n)N(C₀-C₃-alkyl)(n=1, 2), preferably SO₂NH₂ or SO₂NMe₂ or SO_(n)C₁-C₅-alkyl, (n=1, 2)preferably sulphonylmethyl or sulphinylmethyl.

Favoured R⁶(R⁷)(R⁸)-E-configurations include —C(Ph)₃ (trityl), —CH(Ph)₂,—CH₂Ph, —Si(Ph)₂(t-Bu), 1,1-bis(4-methylphenyl)-1′-pyrenylmethyl, wherePh is phenyl or phenyl substituted with R⁴.

Note, however, that the novel compounds of the invention exclude by wayof proviso certain compounds with common protecting groups at the5′-oxygen of the nucleoside, such as 5′-O-trityl, methoxylated5′-O-trityl or 5′-O-tert.butyldiphenylsilyl. Accordingly5′-O-(4′,4′-dimethoxytrityl)-2′-deoxyuridine is outside the scope of thenovel compound aspect of the invention. This exclusion of trityl andtBuPh₂Si in the compound claims only is not believed to be required inrespect of other permutations of C₀-C₃alkyl-D-C₀-C₃ alkyl, such ascompounds wherein D is N. The novel compounds of the invention willhowever typically avoid conventional hydroxyl protecting groups (such asthose cited in Greene below), when C₀-C₃alkyl-D-C₀-C₃ alkyl is —O—CH₂—.It will be appreciated, however, that the use/method of treatmentaspects of the invention include those compounds excluded from thecompound claims by proviso.

Compounds wherein E is carbon are currently favoured on pharmacokineticgrounds, although compounds with E as Si have shown advantageous potencyand selectivity.

The compounds of the invention include a number of chiral centres, andthe invention extends to include racemates, enantiomers andstereoisomers at each of these centres. For example the ring carbonattached to the uracil N1 in Formula I may be in the alpha (down) orpreferably the beta (up) configuration. R₂ as F in Formula I may be inthe ribo (down) position although it is currently preferred to have thearabino (up) position. It is currently preferred that the ring carbonintermediate A and B in Formula I projects the adjacent C₀-C₃ alkylenein the beta configuration.

Compounds of the invention are generally at least 80% preferably atleast 90% such as 97% stereoisometrically pure at chiral centres.

Additional aspects of the invention include a pharmaceutical compositioncomprising a compound of the formula I in conjunction with apharmaceutically acceptable carrier or diluent therefore. The inventionfurther provides a method for the treatment or prophylaxis of parasiteinfections, such as malaria, tuberculosis or leishmaniasis, in man or azoonose vector comprising the administration of an effective amount of acompound of the formula I to a patient in need thereof, or to thevector.

While it is possible for the active agent to be administered alone, itis preferable to present it as part of a pharmaceutical formulation.Such a formulation will comprise the above defined active agent togetherwith one or more acceptable carriers or excipients and optionally othertherapeutic ingredients. The carrier(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the abovedefined active agent with the carrier. In general, the formulations areprepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. The invention extendsto methods for preparing a pharmaceutical composition comprisingbringing a compound of Formula I or its pharmaceutically acceptable saltin conjunction or association with a pharmaceutically acceptable carrieror vehicle. If the manufacture of pharmaceutical formulations involvesintimate mixing of pharmaceutical excipients and the active ingredientin salt form, then it is often preferred to use excipients which arenon-basic in nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may bepresented as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets andcapsules), the term suitable carrier includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example cornstarch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, sodium chloride and alginic acid; andlubricants such as magnesium stearate, sodium stearate and othermetallic stearates, stearic acid, glycerol stearate, silicone fluid,talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring or the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Dosages are set in the conventional manner to take into account theseverity of the disease, the susceptibility of the parasite strain, thesize and metabolic health of the patient, the mode and form ofadministration, concomitant medication and other relevant factors. Thecompounds of the invention may be administered at a daily dose generallyin the range 0.1 to 200 mg/kg/day, advantageously, 0.5 to 100 mg/kg/day,more preferably 10 to 50 mg/kg/day, such as 10 to 25 mg/kg/day. Atypical dosage rate for a normal adult will be around 50 to 500 mg, forexample 300 mg, once or twice per day.

The compounds of formula I can form salts which form an additionalaspect of the invention. Appropriate pharmaceutically acceptable saltsof the compounds of formula I include salts of organic acids, especiallycarboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, isethionate, adipate, alginate, aspartate,benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate,glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate,palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate,tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,organic sulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids.

Examples of monocyclic rings for R¹ include heterocycles such as furyl,thienyl, pyranyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl,pyridyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl,thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, especiallypyridyl, and carbocycles such as cycloalkyl, cycloalkenyl and phenyl.

Examples of monocyclic, bicyclic or tricyclic rings for R⁶, R⁷ and/or R⁸include heterocycles such as furyl, thienyl, pyranyl, pyrrolyl,pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, isothiazolidinyl, thiadiazolyl, tetrazolyl,triazolyl, and the like or bicyclic rings especially of the above fusedto a phenyl ring such as indolyl, quinolyl quinolinyl, isoquinolinyl,benzimidazolyl, benzothiazolyl, benzoxazolyl, benzotriazolyl,benzofuryl, benzothienyl etc. Additional rings include xanthenyl (suchas 9-xanthenyl, 9-alkylxanthenyl, 9-(9-alkyl)xanthenyl,9-phenylxanthenyl, 9-(9-phenyl)xanthenyl, 9-heteroarylxanthenyl,9-(9-heteroaryl)xanthenyl), dibenzosuberyl, 5-dibenzosuberyl, fluorenyl(such as 5-fluorenyl, 5-(5-alkyl)fluorenyl, 5-(5-phenyl)fluorenyl,5-(5-heteroaryl)fluorenyl) and the like.

Examples of monocyclic, bicyclic or tricyclic ring systems with anaromatic nature for R⁶, and/or R⁷ include heteroaryls such as furyl,thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,thiadiazolyl, tetrazolyl, triazolyl, and the like or bicyclic ringsespecially of the above fused to a phenyl ring such as indolyl,quinolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl,benzoxazolyl, benzotriazolyl, benzofuryl, benzothienyl etc. Additionalrings include xanthenyl (such as 9-xanthenyl, 9-alkylxanthenyl,9-(9-alkyl)xanthenyl, 9-phenylxanthenyl, 9-(9-phenyl)xanthenyl,9-heteroarylxanthenyl, 9-(9-heteroaryl)xanthenyl), dibenzosuberyl,5-dibenzosuberyl, fluorenyl (such as 5-fluorenyl, 5-(5-alkyl)fluorenyl,5-(5-phenyl)fluorenyl, 5-(5-heteroaryl)fluorenyl) and the like.

Examples of carbocycles for R⁶, R⁷ and/or R⁸ include monocyclic ringssuch as phenyl, cyclohexenyl, cyclopentenyl, cyclohexanyl,cyclopentanyl, bicyclic rings such as indanyl, napthyl, and tricyclicrings such as adamantyl, and the like.

The carbo or heterocyclic ring may be bonded via a carbon or via ahetero atom, typically a nitrogen atom, such as N-piperidyl,N-morpholinyl etc. Other examples of such ring systems may also be foundin J. Fletcher, O. Dermer, R. Fox, Nomenclature of Organic Compounds,pp. 20-63 (1974).

The term “C₁-C₅ alkyl” includes such groups as methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl, t-butyl, cyclopropyl, n-pentyl and the likewith C₁-C₈ alkyl further including n-hexyl, 3-methylpentyl, and thelike.

The term “halo” and “halogen” refer to chloro, bromo, iodo, andespecially fluoro.

“C₁-C₅ alkoxy” refers to those groups such as methoxy, ethoxy, propoxy,t-butoxy and the like.

“C₂-C₅ alkenyl” refers to those groups such as vinyl, 1-propen-2-yl,1-butene-4-yl, 1-pentene-5-yl, 1-butene-1-yl and the like, with C₂-C₈alkenyl further including hex-3-enyl and the like.

“C₁-C₅ alkylthio” refers to those groups such as methylthio, ethylthio,t-butylthio, and the like.

“C₁-C₅ alkanoyl” refers to groups such as acetyl, propionyl, butyryl andthe like.

“C₁-C₅ alkanoyloxy” refers to those groups such as acetoxy, propionoxy,formyloxy, butyryloxy, and the like.

The term “C₂-C₈ alkenoxy” includes groups such as ethenyloxy,propenyloxy, iso-butoxy ethenyl, and the like.

The term “C₂-C₅ alkynyl” includes groups such as ethynyl, propynyl,butynyl, pentynyl, and the like with C₂-C₈ alkynyl further includinghexynyl and the like.

The term “halo C₁-C₅ alkyl” includes alkyls substituted 1, 2 or 3 timesby a halogen including groups such as trifluoromethyl, fluoromethyl,2-dichloroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl,3,3-difluoropropyl, 1,1-2,2,2 pentafluoroethyl and the like.

The term —C₀-C₃-alkylene- as a bivalent in expressions such as—C₀-C₃-alkylene-D-C₀-C₃-alkylene includes a bond (i.e C₀), methylene(C₁), ethylene (C₂), 1,1-dimethyl-methylene (C₃), propylene (C₃) and thelike, with each —C₀-C₃-alkylene- being selected independently.

The term (C₀-C₃-alkyl) in monovalent expressions includes H (i.e C₀), Me(C₁), Et (C₂), propyl (C3) with each C₀-C₃-alkyl being selectedindependently. Accordingly —N(C₀-C₃-alkyl)₂ includes —NH₂, —NHMe, NHEtNHPr, —N(Me)₂, N(Et)₂ etc, —SO₂N(C₀-C₃-alkyl)₂, includes —SO₂NH₂,—SO₂NHMe, —SO₂N(Me)₂ etc

As used herein, “the esters, amides and ethers thereof” refer to theappropriate derivatives of each of the preceding hydroxyl or aminogroups in the respective definition.

Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C₁₋₄ alkyl, or C₁₋₄ alkoxy) or amino); sulphonateesters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); amino acid esters (for example, L-valyl orL-isoleucyl); and mono-, di-, or tri-phosphate esters. In such esters,unless otherwise specified, any alkyl moiety present advantageouslycontains from 1 to 18 carbon atoms, particularly from 1 to 6 carbonatoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moietypresent in such esters advantageously contains from 3 to 6 carbon atoms.Any aryl moiety present in such esters advantageously comprises anoptionally R⁴-substituted phenyl group.

Pharmaceutically acceptable esters thus include C₁-C₂₂ fatty acidesters, such as acetyl, t-butyl or long chain straight or branchedunsaturated or omega-6 monounsaturated fatty acids such as palmoyl,stearoyl and the like.

Alternative aryl or heteroaryl esters include benzoyl, pyridylmethyloyland the like any of which may be substituted with R⁴, Preferredpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl. Additional preferredamino acid esters include the 2-O-AA-C₃-C₂₂ fatty acid esters describedin WO99 09031, where AA is an aliphatic amino acid ester, especiallythose derived from L-lactic acid and L-valyl.

Pharmaceutically acceptable ethers include straight or branched chainsaturated or omega 6 unsaturated C₁-C₂₂ alkyl ethers such as methylethers, t-butyl ethers or aryl or heteroaryl ethers such as phenoxy,benzylether, pyridylmethyl ether, any of which may be substituted withR⁴.

Alternative ethers include alkylated silyl functions such as—Si(C₁-C₅-alkyl)₃ such as —Si(t-Bu)(CH₃)₂, or —Si(Ph)₂(t-Bu), C(Ph)₃(trityl), —CH(Ph)₂, —CH₂Ph,1,1-bis(4-methylphenyl)-1′-pyrenylmethyl andthe like.

Pharmaceutically acceptable amides include those derived from C₁-C₂₂branched or straight chain aminoalkyl optionally including 1 to 3unsaturations and/or optionally substituted with R⁴, or anilines orbenzylamines. Preferred amides include those formed from reaction of theamine with a C₁-C₄ straight or branched chain alkanoic acid. Otherpharmaceutically acceptable amides of amine functions of R² or R¹¹correspond to the esters indicated above.

It is currently preferred that the ester, amide or ether is lipophilicin nature.

Compounds of the invention are typically synthesized as outlined below.

Scheme 1 depicts a method for alkylation of the 5′-position of anucleoside or a nucleoside analogue.

Nucleoside derivative (1a) wherein A, B, R¹ and R² are as defined abovefor formula I and D is O or NH, can be reacted with an alkylating agentof formula Ib wherein R⁶, R⁷, R⁸ and E are as defined above for formulaI and Lg is a leaving group that can be replaced by the nucleophile D,in a solvent like pyridine optionally in the presence of a catalyst suchas dimethylaminopyridine or in a solvent like dimethylformamide in thepresence of a catalyst like imidazole, to provide 5′-alkylatednucleoside analogues (1c). Various alkylating agents (1b) are availableeither commercially or in the literature, se for example Greene,“Protective Groups in Organic Synthesis (John Wiley & Sons, New York,1981). For example, they can be prepared by transforming the hydroxygroup of the corresponding alcohol into a leaving group such as a halidelike chloride or bromide by treatment with a halogenating agent such asacetyl bromide or thionyl chloride or the like or they can betransformed into a derivative of sulfonic acid like a mesyl, tosyl,triflate or the like by treatment with for example the anhydride or acidchloride of the desired sulfonic acid derivative. An example of a routeto alkylating agents is shown in scheme 2.

Reaction of an electrophilic carbonyl compound like a keto compound (2a)or any carboxylic acid derivative for instance an ester or acid halide,and a suitable nucleophile for example a Grignard reagent (2b) or anorganolithium reagent, provides the alcohol (2c). The formed hydroxygroup can subsequently be transformed into a leaving group as describedabove thus forming the alkylating agent (1b). Examples of the aboveprocedure are described in the literature, se for example Hodges et al.,J. Org. Chem. 56, 1991, 449-452, and Jones et al., J. Med. Chem. 33,1990, 416-419.

Compounds wherein the leaving group in the alkylating agent (1b) isspaced by a C₁-C₃-alkylene chain, available either commercially or inthe literature, may also be used as alkylating agents in scheme 1. Anexample of a route to a compound containing a C₂-alkylene chain is shownin scheme 3.

A reaction performed with triphenylmethyl sodium (3a) and ethylene oxideprovides alcohol (3b). Subsequent transformation of the hydroxy groupinto a leaving group for example as described above provides alkylatingagent (3c). Use of any other appropriate electrophilic reagent forexample formaldehyde, provides analogues with other length of theC₁-C₃-alkyl chain. Se for example Wooster et al., J. Amer. Chem. Soc.,60, 1938, 1666 and McPhee et al., J. Amer. Chem. Soc. 65, 1943, 2177,2180. Alternatively, alkylating agents containing a C₁-C₃-alkyl chainmay be obtained by reduction of an appropriate acyl derivative to thedesired alcohol.

A suitable acylating agent like the acid chloride or anhydride can beused to acylate the amino group of a 5′-aminonucleoside, thus providingcompounds according to the general formula I where D is —CONH—.

The 5′-substituent can also be introduced by way of a Mitsunobu reactionof a desired alcohol and the 5′-unprotected nucleoside derivative asillustrated in scheme 3A.

Treatment of a desired optionally suitably protected alcohol (3Aa) and anucleoside derivative (3Ab) with triphenyl phosphine and DIAD in asolvent like THF provides the nucleoside analogue (3Ac).

An example of the introduction of an ether group at the 3′-position ofthe nucleoside analogue is shown in scheme 4.

Treatment of a 5′-substituted nucleoside analogue (4a) with a silylatingagent for example tert-butyldimethylsilyl chloride in a solvent likedimethylformamide in the presence of a catalyst like imidazole, provides3′-O-silylated derivatives (4b).

Other ether or ester groups can be introduced at the 3′-position bymethods known in the art, for example by treating the 3′-OH nucleosidewith the desired alkylating or acylating agent optionally in thepresence of a suitable base, se for example Greene, “Protective Groupsin Organic Synthesis (John Wiley & Sons, New York, 1981).

Nucleoside analogues used in the synthesis of compounds according to thepresent invention are available either commercially or in the literatureor they can be prepared as described herein. For example compound 1wherein B is CH₂F, R¹ and R² are H and A and D are O i.e. FLU(3′-fluoro-2′,3′-dideoxyuridine) can be prepared in analogy with theprocedure described for FLT (Balzarini et al. BIochem. Pharmacol. 37,2847, 1988). The didehydro derivative d4U(2′,3′-didehydro-2′,3′-dideoxyuridine) can be prepared in analogy withthe procedure described for d4T (2′,3′-didehydro-2′,3′-dideoxythymidine,Stavudine, Balzarini et al.; Mol. Pharmacol. 32, 162, 1987).2′-Fluoro-2′deoxyarabino furanosyluracil is conveniently prepared forexample as described by H. Howell in J. Org. Chem., 53, 85, 1988 and thecorresponding ribo derivative, 2′-fluoro-2′deoxyribofuranosyluracil canbe prepared as described for example by Mercer et al. in J. Med. Chem.,30, 670-675, 1987.

5′-aminonucleoside analogues, useful for the preparation of compoundsaccording to the general formula I wherein D is NH or —CONH— can beprepared from the corresponding 5′-alcohols by a displacement-reductionsequence for example as shown in scheme 5.

Nucleoside analogue 5a where Y is F or suitably protected OH or NH₂, canbe reacted with triphenylphosphine in a solvent like carbon tetrabromidefollowed by displacement of triphenylphosphine oxide with azide ion toform 5b. Alternatively the hydroxy group can be transformed into aleaving group such as a halide like chloride or bromide or a derivativeof sulphonic acid such as a tosyl, mesyl or a triflyl group which issubsequently displaced by azide ion. Reduction of the azide group forexample by catalytic hydrogenation over palladium on carbon, gives theamino derivative 5c.

The procedures described in scheme 5 can be also applied to carbocyclicuridine and thiouridine analogues providing compounds useful for thepreparation of compounds of the general formula I wherein A is CH₂ andS.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various aspects of the invention, including end product inhibitors andintermediates towards those inhibitors will now be described by way ofillustration only with reference to the following non-limiting examples.Note that the exemplified intermediates, such as the acyclic side chainbuilding blocks are readily reacted with alternative bases to formadditional compounds of the invention.

Example 1

1-(5-Trityloxymethyl-2,5-dihydro-furan-2-yl)-1H-pyrimidine-2,4-dione or5′-O-trityl-2′,3′-dideoxydidehydrouridine (1)

2′,3′-Dideoxy-didehydrouridine (0.30 g, 1.43 mmol) and triphenylmethylchloride (0.44 g, 1.57 mmol) was stirred in dry pyridine (10 ml) at 50°C. overnight under an atmosphere of nitrogen. The reaction mixture waspoured into ice-H₂O (30 ml) with vigorous stirring and filtered. Theprecipitate was dissolved in EtOAc (50 ml) and the solution was washedwith 0.5M HCl (20 ml), H₂O (20 ml), dried (Na₂SO₄) and reduced in vacuoto yield a crude product, which was purified by gradient flash columnchromatography eluting with 0→3% MeOH/CHCl₃ to yield the title compoundas white crystals (0.37 g, 58%).

¹H NMR (300 MHz; MeOH): δ 3.56 (2H, m, 5′-H), 5.02 (1H, m, 4′-H), 5.08(1H, m, 5-H), 5.93 (1H, m, 1′-H), 6.40 (1H, m, 2′-H), 7.09 (1H, m,3′-H), 7.30-7.44 (15H, m, Ph-H), 7.87 (1H, d, J=8.1 Hz, 6-H);

¹³C NMR (75 MHz; MeOH): δ 64.82 (5′-CH₂), 86.38 (1′-CH), 87.84 (a),90.04 (4′-CH), 102.70 (5-CH), 126.79 (Ph-CH), 127.79 (2′-CH), 128.41(Ph-CH), 129.20 (Ph-CH), 134.89 (3′-CH), 141.79 (6-CH), 143.49 (Ph-C),151.04 (2-C), 159.95 (4-C);

MS (Cl/NH₃, m/z); 470.2 (M+NH₄ ⁺, 100%), 453.1 (M+H⁺, 20%);

HRMS (ES+ve., M+H): Calculated for C₂₈H₂₄N₂O₄, requires 453.1814, found453.1807.

IR_(vmax)/cm⁻¹ (KBr): 714 and 756 (Aromatic, monosubstituted), 1681.0(C═O) and 1692.3 (C═O); Mp: 68° C., R_(f) (10% MeOH/CHCl₃): 0.30;

Example 2

5′-O-tert-butyldiphenylsilyl-2′,3′-dideoxydidehydrouridine (2)

2′3′-dideoxy-didehydrouridine (0.30 g, 1.43 mmol) in dry DMF (10 ml)were added drop-wise under an atmosphere of nitrogen, with ice bathcooling, to a stirred solution of tert-butyldiphenyllsilylchloride (0.41ml, 1.57 mmol) and imidazole (0.21 g, 3.14 mmol) in dry DMF (10 ml). Themixture was allowed to warm to room temperature and stirred overnight.

H₂O (10 ml) was added and the mixture was extracted with CHCl₃ (2×30ml). The combined extracts were washed with saturated aqueous NaHCO₃solution (10 ml) and H₂O (10 ml), dried (Na₂SO₄) and reduced in vacuo toobtain a crude product, which was purified by gradient flash columnchromatography eluting with 0→3% MeOH/CHCl₃ to yield the title compoundas colourless viscous oil (0.46 g, 73%).

¹H NMR (300 MHz; MeOH): δ 1.15 [9H, s, C(CH₃)₃], 3.95 (1H, dd, J=2.9,11.7 Hz, 5′-H), 4.06 (1H, dd, J=2.9, 11.7 Hz, 5′-H), 4.97 (1H, m, 4′-H),5.26 (1H, d, J=8.1 Hz, 5-H), 5.58 (1H, m, 1′-H), 6.38 (1H, m, 2′-H),7.10 (1H, m, 3′-H), 7.34-7.55 (6H, m, Ph-CH), 7.66-7.81 (5H, m, Ph-H and6-H).

¹³C NMR (75 MHz; MeOH): δ 19.81 [C(CH₃)₃], 26.99 and 27.42 [C(CH₃)₃],65.40 (5′-CH₂), 87.56 (1′-CH), 90.06 (4′-CH), 102.96 (5-CH), 126.96(2′-CH), 128.41 (Ph-CH), 128.32 (Ph-CH), 128.12 (Ph-CH), 130.03 (Ph-CH),130.47 (Ph-CH), 130.59 (Ph-CH), 132.78 (Ph-C), 133.46 (Ph-C), 134.99(3′-CH), 135.25 (Ph-CH), 135.79 (Ph-CH) 135.99 (Ph-CH), 141.20 (6-CH),150.99 (2-C), 163.45 (4-C);

MS (Cl/NH₃., m/z); 449.1 (M+H⁺, 50%), 466.2 (M+NH₄ ⁺, 100%);

HRMS (ES+ve., M+H⁺): Calculated for C₂₅H₂₈N₂O₄Si, requires 449.1896,found 449.1894.

IR_(vmax)/cm⁻¹ (film): 1697.3 (C═O).

R_(f) (10% MeOH/CHCl₃): 0.73.

Example 3

Diphenyl (pyridin-3-yl)methanol (3)

A solution of 3-bromopyridine (10 g, 0.063 mol) in dry THF (200mL)/hexane (50 mL) was cooled to −90° C. To this cooled solution wasadded n-BuLi (2.2 M, 32 mL, 0.063 mol) slowly and allowed to stir for 30min under N₂ atmosphere. A solution of benzophenone (11.5 g, 0.063 mol)in dry THF (50 mL) was added to this at the same temperature over aperiod of 30 min. The reaction mixture was warmed slowly to RT andallowed to stir another 3 h at RT. The reaction mixture was cooled,quenched with water (200 mL) and extracted with ethylacetate (2×100 mL).The organic layer was dried, concentrated under vacuum and crudepurified by column chromatography over silica gel (30% ethyl acetate inpetroleum ether) to give the title product (3.3 g).

Example 4

5′-O-Tosyl-2′-deoxyuridine (4)

To an ice-cold solution of 2′-deoxyuridine (5 g, 0.0219 mol) in drypyridine (25 mL) tosyl chloride (5 g, 0.0263 mol) was added portion wisewith stirring. The reaction mixture was stirred at 0° C. for 12 h. Thereaction mixture was concentrated under vacuum and the crude residue waswashed with diethyl ether (5×25 mL). The residue was further treatedwith water. The solid precipitate formed was filtered, washed with water(2×25 mL), diethyl ether (5×25 mL) and petroleum ether (5×25 mL). Thesolid was dried under vacuum and used for next reaction without anypurification. Yield: 7.5 g, 89%.

TLC: CHCl₃/MeOH, 4:1, R_(f)=0.6

Example 5

5′-Azido-2′,5′-dideoxyuridine (5)

To a solution of 5′-O-tosyl-2′-deoxyuridine (13.5 g, 0.035 mol) in dryDMF (90 mL) was added NaN₃ (9.2 g, 0.141 mol) and the reaction mixturewas allowed to stir at 95° C. for 12 h. The reaction mixture was cooled,the solid residue was removed by filtration and the filtrate wasconcentrated under vacuum to give the crude product. The crude waspurified by column chromatography on silica gel (4% methanol inchloroform) which gave the title product (5.2 g, 56%) as a white solid.

TLC: CHCl₃/MeOH, 4:1, R_(f)=0.45

Example 6

5′-Amino-2′,5′-dideoxyuridine (6)

To a mixture of 5′-azido-2′,5′-dideoxyuridine (5 g, 0.0197 mol) inmethanol/water (150 mL, 1:1) was added Pd/C (0.25 g, 10%) under N₂atmosphere and then hydrogenated for 4 h at RT. The reaction mixture wasfiltered through bed of celite and the filtrate was concentrated undervacuum. The solid obtained was washed with 3% methanol in chloroformwhich gave the title product (4.1 g, 89%) as an off-white solid.

TLC: CHCl₃/MeOH, 4:1, R_(f)=0.1.

Example 7

5′-O-Triisopropylsilyl-2′-deoxyuridine (7)

Imidazole (0.183 g, 2.69 mmol) was added to a solution of2′-deoxyuridine (0.272 g, 1.19 mmol) in dry DMF (5 mL) under nitrogen.The mixture was cooled in an ice-salt bath before drop-wise addition oftriisopropylsilyl chloride (0.28 mL, 1.31 mmol) via a syringe. Thereaction mixture was kept at 0° C. for 3 h, allowed to warm up to roomtemperature and then stirred at room temperature for 22 h. Afteraddition of water (5 mL), the crude mixture was extracted with CHCl₃(2×10 mL). The organic layers were combined and dried over MgSO₄.Removal of the solvent under reduced pressure afforded a crudetransparent oil which was further purified by flash chromatographyeluting the column (ISOLUTE SI) a gradient of 0→10% CH₃OH in CHCl₃. Thetitle compound was obtained from the fractions with R_(f)=0.25 (10%CH₃OH in CHCl₃) as a crystalline white solid (0.366 g, 74%). M.p.152-153° C.

¹H NMR (300 MHz, CDCl₃) δ 1.08 (21H, m, Pr-H), 2.18 (1H, m, 2′-H), 2.49(1H, m, 2′-H), 3.97 (2H, m, 5′-H), 4.08 (1H, m, 4′-H), 4.56 (1H, m,3′-H), 5.70 (1H, d, J=8.1 Hz, 5-H), 6.38 (1H, t, J=6.2 Hz, 1′-H), 7.96(1H, d, J=8.1 Hz, 6-H), 10.16 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 12.7 (iPr-CH), 18.4 (iPr-CH₃), 41.9 (2′-CH₂),63.8 (5′-CH₂), 71.7 (3′-CH), 85.7 (1′-CH), 88.0 (4′-CH), 102.5 (5-CH),140.9 (6-CH), 151.1 (2-C), 164.5 (4-C).

ES⁺ m/z (%) 790 ([2M+Na]⁺, 10), 407 ([M+Na]⁺, 100).

HRMS (ES⁺) Found [M+Na]⁺407.1988; C₁₈H₃₂N₂O₅SiNa⁺ requires 407.1973.

Anal. Calcd for C₁₈H₃₂N₂O₅Si (%) C, 56.22; H, 8.39; N, 7.28, found C,56.07; H, 8.50; N, 7.18.

Example 8

5′-(4-pyridydiphenylmethyl)amino-2′,5′-dideoxyuridine (8)

5′-Amino-2′,5′-dideoxyuridine (0.108 g, 0.475 mmol) was added to asolution of diphenyl(4-pyridyl)chloromethane hydrochloride (0.150 g,0.474 mmol), pyridine (3 mL) and Et₃N (0.12 mL, 0.862 mmol). Thereaction mixture was heated at 40° C. for 4 h then the temperature wasincreased to 70° C. for 10 h. The crude solution was partitioned betweenwater (5 mL) and EtOAc (3×7 mL). The organic extracts were combined,dried over Na₂SO₄ and concentrated in vacuo. The brown solid obtainedwas taken in MeOH and the remaining insoluble material was filtered off.The filtrate was concentrated under reduced pressure and furtherpurified by flash column chromatography (ISOLUTE SI column) using agradient elution of 0→8% MeOH in CHCl₃. The fractions with R_(f)=0.24(10% MeOH/CHCl₃) afforded the title compound as a pale yellow solid (61mg, 27%).

¹H NMR (300 MHz, CD₃OD) δ 2.15-2.60 (4H, m, 2′,5′-H), 4.03 (1H, m, 3′-Hor 4′-H), 4.18 (1H, m, 3′-H or 4′-H), 5.63 (1H, d, J=8.0 Hz, 5-H), 6.21(1H, t, J=6.4 Hz, 1′-H), 7.20-7.48 (11H, m, 6-H and Ph-H), 7.59 (2H, d,J=5.9 Hz, 4″-H), 8.42 (2H, d, J=5.9 Hz, 5″-H).

¹³C NMR (75 MHz, CD₃OD) δ 40.9 (2′-CH₂), 47.6 (5′-CH₂), 72.2 (2″-C),73.3 (3′-CH), 87.1 (1′-CH or 4′-CH), 87.9 (1′-CH or 4′-CH), 103.4(5-CH), 125.7 (4″-CH), 128.6 (Ph-CH), 129.7 (Ph-CH), 130.2 (Ph-CH),130.3 (Ph-CH), 142.7 (6-CH), 145.8 (Ph-C), 146.1 (Ph-C), 150.2 (5″-CH),152.4 (3″-C), 158.1 (2-C), 166.4 (4-C).

ES⁺ m/z (%) 963 ([2M+Na]⁺, 13), 493 ([M+Na]⁺, 84), 471 ([M+H]⁺ , 13).

HRMS (ES⁺) Found [M+H]⁺ 471.2033; C₂₇H₂₇N₄O₄ ⁺ requires 471.2027.

M.p. 131-133° C.

Example 9

5′-O-trityl-2′-deoxyuridine (9)

2′-Deoxyuridine (1.00 g, 4.39 mmol) and triphenylmethylchloride (1.34 g,4.83 mmol) were stirred in dry pyridine (20 ml) overnight at 50° C.under an atmosphere of nitrogen. The reaction mixture was then pouredinto ice-H₂O (100 ml) with vigorous stirring and filtered. Theprecipitate was dissolved in EtOAc (100 ml) and the solution was washedwith 0.5M HCl (100 ml) and H₂O (100 ml), dried (Na₂SO₄) and reduced invacuo. The residue was washed with toluene to leave the title compound(1.99 g, 97%) as a pale yellow solid. For analytical purposes, thecompound was purified by gradient flash column chromatography, elutingwith 5→10% MeOH/CHCl₃.

¹H NMR (300 MHz; CDCl₃): δ 2.34 (1H, m, 2′-H), 2.45 (1H, m, 2′-H), 3.51(2H, ddd, J=3.5, 8.6, 10.6 Hz, 5′-H), 4.12 (1H, dd, J=3.6, 7.2 Hz,4′-H), 4.64 (1H, m, 3′-H), 5.47 (1H, d, J=8.1 Hz, 5-H), 6.40 (1H, t,J=6.3 Hz, 1′H), 7.22-7.49 (15H, m, Ph-H), 7.86 (1H, d, J=8.1 Hz, 6-H),9.37 (1H, s, 3—NH);

¹³C NMR (75 MHz; CDCl₃): δ 41.60 (2′-CH₂), 63.53 (5′-CH₂), 71.84(3′-CH), 85.49 (4′-CH), 86.43 (1′-CH), 88.03 (Ph₃C), 127.92 (Ph-CH),128.68 (Ph-CH), 129.49 (Ph-CH), 140.69 (6-CH), 143.67 (Ph-CH), 153.24(2-C), 163.93 (4-C).

MS (AP⁺., m/z): 243 (Tr⁺, 100%); R_(f) (10% MeOH/CHCl₃): 0.49;

Example 10

1-[4-(tert-Butyl-dimethyl-silanyloxy)-5-trityloxymethyl-tetrahydro-furan-2-yl]-1Hpyrimidine-2,4-dione or 3′-O-tert-Butylsilyl-5′-O-trityl-2′-deoxyuridine(10)

5′-O-trityl-2′deoxyuridine (0.70 g, 1.49 mmol) in dry DMF (3 ml) wasadded drop-wise under an atmosphere of nitrogen, with ice bath cooling,to a stirred solution of tert-butyldimethylsilyl chloride (0.25 g, 1.65mmol) and imidazole (0.22 g, 3.28 mmol) in dry DMF (3 ml). The mixturewas allowed to warm to room temperature and stirred overnight. H₂O (10ml) was added (10 ml) and the mixture was extracted with Et₂O (2×50 ml).The combined extracts were washed with saturated NaHCO₃ (50 ml) and H₂O(50 ml), dried (Na₂SO₄) and reduced in vacuo. A flash silica columneluting with 3% MeOH/CHCl₃ gave the title compound (0.65 g, 74%) aswhite foam.

¹H NMR (300 MHz; CDCl₃): δ-0.05 [3H, s, Si(CH ₃)₂] and 0.00 [3H, s,Si(CH ₃)₂], 0.85 [9H, s, C(CH ₃)₃], 2.12-2.20 (1H, m, 2′-H), 2.31-2.39(1H, m, 2′-H), 3.33 (1H, dd, J=2.8, 10.7 Hz, 5′-H), 3.46 (1H, dd, J=2.9,10.7 Hz, 5′-H), 3.92 (1H, dt, J=2.8, 4.4 Hz, 4′-H), 4.51 (1H, dd, J=4.9,10.9 Hz, 3′-H), 5.34 (1H, d, J=8.1 Hz, 5-H), 6.26 (1H, t, J=6.0 Hz,1′-H), 7.23-7.39 (15H, m, Ph-H), 7.85 (1H, d, J=8.1 Hz, 6-H), 9.11 (1H,s, 3—NH);

¹³C NMR (75 MHz; CDCl₃): −4.49 and −4.20 (Si(CH₃)₂), 18.37[C(CH₃)₃],22.06 and 26.17 [C(CH₃)₃], 42.23 (2′-CH₂), 62.27 (5′-CH₂), 71.38(3′-CH), 85.55 (4′-CH) 86.83 (1′-CH), 87.89 (Ph₃C), 102.70 (5-CH),127.91 (Ph-CH), 128.48 (Ph-CH), 129.15 (Ph-CH), 140.62 (6-CH), 143.59(Ph-CH), 150.68 (2-C), 163.81 (4-C);

MS (AP⁺., m/z): 243 (Tr⁺, 50%), 341 (M−Tr⁺, 75%), 607 (M+Na⁺, 100%)

R_(f) (3% MeOH/CHCl₃): 0.33;

Example 11

5′-O-Triphenylsilyl-2′-deoxyuridine (11)

A solution of triphenylsilyl chloride (0.437 g, 1.48 mmol) in drypyridine (4 mL) was added drop-wise to a solution of 2′-deoxyuridine(0.278 g, 1.22 mmol) in dry pyridine (4 mL) previously cooled in anice-salt bath. The reaction mixture was kept at 0° C. for 1 h. Thereaction was monitored by TLC (10% CH₃OH in CHCl₃) and quenched withCH₃OH (50

L). The solvent was removed under reduced pressure to give a crudeyellow liquid which was further purified by silica gel columnchromatography (Isolute SI column) using a gradient elution of 0→10%CH₃OH in CHCl₃. The fractions with R_(f)=0.30 (10% CH₃OH/CHCl₃) werecombined and concentrated to yield the title compound as a whitecrystalline solid (0.506 g, 85%).

¹H NMR (300 MHz, CDCl₃) δ 2.25 (1H, m, 2′-H), 2.44 (1H, m, 2′-H), 2.95(1H, bs, 3′-OH), 3.93-4.27 (3H, m, 5′-H and 4′-H), 4.60 (1H, m, 3′-H),5.19 (1H, d, J=8.2 Hz, 5-H), 6.41 (1H, t, J=6.4 Hz, 1′-H), 7.35-7.73(15H, m, Ph-H), 7.80 (1H, d, J=8.1 Hz, 6-H), 9.46 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 41.6 (2′-CH₂), 63.8 (5′-CH₂), 71.7 (3′-CH),85.3 (1′-CH), 87.3 (4′-CH), 102.7 (5-CH), 128.6 (Ph-CH), 131.1 (Ph-CH),133.3 (Ph-C), 135.8 (Ph-CH), 140.5 (6-CH), 150.9 (2-C), 163.9 (4-C).

ES⁺ m/z (%) 509 ([M+Na]⁺, 100).

ES⁺ m/z (%) 509 ([M+Na]⁺, 78), 151 (100).

HRMS (ES⁺) Found [M+Na]⁺509.1504; C₂₇H₂₆N₂O₅Si requires 509.1503.

Anal. calcd for C₂₇H₂₆N₂O₅Si (%): 0.32 HCl C, 65.09; H, 5.32; N, 5.62found: C, 65.01; H, 5.27; N, 5.62.

Example 12

5′-O-tert-Butyldiphenylsilyl-2′-deoxyuridine (12)

2′-Deoxyuridine (0.530 g, 2.32 mmol) was dissolved in dry DMF (5 mL)under nitrogen and the solution was cooled in an ice-salt bath. Asolution of tert-butyldiphenylsilyl chloride (0.710 g, 2.58 mmol) andimidazole (0.342 g, 5.69 mmol) in dry DMF (4 mL) was then addeddrop-wise. The reaction mixture was stirred at 0° C. for 2 h and then atroom temperature for 15 h. The reaction was quenched by addition ofwater (15 mL). The crude mixture was extracted with CHCl₃ (2×15 mL). Theorganic layers were combined, dried over MgSO₄ and concentrated in vacuoto give a transparent oil (0.419 g). This oil was chromatographed on asilica gel column (Isolute SI column) eluted with a gradient of 0→10%CH₃OH in CHCl₃. The fractions with R_(f)=0.26 (10% CH₃OH/CHCl₃) weregathered and concentrated to afford the title compound as a whitecrystalline solid (0.823 g, 76%).

¹H NMR (300 MHz, CDCl₃) δ 1.14 (9H, m, tBu-H), 2.27 (1H, m, 2′-H), 2.50(1H, m, 2′-H), 2.69 (1H, bs, 3′-OH), 3.90 (1H, m, 4′-H), 4.05 (2H, m,5′-H), 4.60 (1H, m, 3′-H), 5.52 (1H, d, J=8.1 Hz, 5-H), 6.41 (1H, t,J=6.4 Hz, 1′-H), 7.48 (6H, m, Ph-H), 7.70 (4H, m, Ph-H), 7.87 (1H, d,J=8.1 Hz, 6-H), 9.34 (1H, bs, 3—NH).

¹³C NMR (75 MHz, CDCl₃) δ 19.7 (tBu-C), 27.4 (tBu-CH₃), 41.7 (2′-CH₂),64.1 (5′-CH₂), 71.9 (3′-CH), 85.4 (1′-CH), 87.5 (4′-CH), 102.6 (5-CH),128.4 (Ph-CH), 128.5 (Ph-CH), 130.6 (Ph-CH), 132.7 (Ph-C), 133.1 (Ph-C),135.8 (Ph-CH), 136.0 (Ph-CH), 140.5 (6-CH), 150.9 (2-C), 163.9 (4-C).

ES⁺ m/z (%) 489 ([M+Na]⁺, 100).

Anal. calcd for C₂₅H₃₀N₂O₅Si 0.58 (%): HCl C, 61.56; H, 6.32; N, 5.74,found C, 61.61; H, 6.23; N, 5.72.

Example 13

3′,5′-O-bistertbutyldimethylsilyl-2′-deoxyuridine

A solution of t-butyl dimethylsilylchloride (2.18 g, 14.46 mmol) andimidazole (1.07 g, 28.92 mmol) in DMF (30 ml) was added slowly (dropwise) to a stirred solution of 2′-deoxyuridine (3 g, 13.15 mmol) in dryDMF (40 ml), with ice-bath cooling at 0° C., under atmosphere ofnitrogen.

After 2 hours, H₂O (100 ml) was added and the mixture was extracted withAcOEt (3×100 ml). The combined extracts were washed with saturatedNaHCO₃ (2×100 ml), dried (MgSO₄) and concentrated. The residue waspurified by flash chromatography and the title compound was isolated asa white amorphous solid from the fractions with Rf=0.65 (10% CH₃OH inCHCl₃).

¹H-NMR (300 MHz, CDCl₃) δ 0.2 (s, 12H, tBu[CH₃]₂Si), 1.0 (s, 18H,tBu[CH₃]₂Si), 2.5 (1H, m, 2′-H), 2.2 (1H, m, 2′-H), 3.06 (1H, d, J=5.0Hz, 3′-H), 3.95 (1H, dd, J=11.5 Hz, 2.2 Hz, 5′-H), 4.01 (1H, dd, J=11.5,2.6 Hz, 5′-H), 4.15 (1H, m, 4′-H), 5.78 (1H, d, J=8.23 Hz, 5-H), 6.45(1H, t, J=6.95 Hz, 1′-H), 8.02 (1H, d, J=8.2 Hz, 6-H), 9.5 (1H, s,3—NH).

¹³C-NMR (75 MHz, CDCl₃) δ 163.9 (4-C), 150.9 (2-C), 140.8 (6-CH), 102.7(5-CH), 87.9 (4′-CH), 85.8 (1′-CH), 72.5 (3′-CH), 63.7 (5′-CH₂), 42.0(2′-CH₂), 26.3 (CH₃), 18.8 (CH₃).

Example 14

5′-Tritylamino-2′,5′-dideoxyuridine (14)

5′-Amino-2′,5′-dideoxyuridine (0.200 g, 0.88 mmol) was taken in drypyridine (5 mL) and the mixture was sonicated for a few minutes. Tritylchloride (0.278 g, 1.00 mmol) was added and the reaction mixture wasstirred at 50° C. overnight. The reaction was then quenched with water(20 mL). The crude mixture was extracted with DCM (3×10 mL). The organiclayers were combined, washed with water (10 mL), dried over MgSO₄ andconcentrated on the rotary evaporator. The resultant brown oil wasfurther purified by silica gel column chromatography (Isolute SI column)using a gradient elution of 0→10% CH₃OH in CHCl₃. The fractions withR_(f)=0.28 (10% CH₃OH/CHCl₃) were pooled and evaporated to dryness toyield the title compound as a white solid (0.202 g, 49%).

¹H NMR (300 MHz, CDCl₃) δ 2.07 (2H, m, 2′-H), 2.28-2.53 (2H, m, 5′-H),2.73 (1H, dd, J=3.5, 12.1 Hz, 1″—NH), 2.97 (1H, bs, 3′-OH), 4.19 (1H, m,4′-H), 4.33 (1H, m, 3′-H), 5.72 (1H, d, J=8.1 Hz, 5-H), 6.36 (1H, t,J=6.4 Hz, 1′-H), 7.14 (1H, d, J=8.1 Hz, 6-H), 7.23-7.43 (9H, m, Ph-H),7.57 (6H, m, Ph-CH), 9.47 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 40.8 (2′-CH₂), 46.6 (5′-CH₂), 71.1 (2″-C),73.0 (3′-CH), 85.4 (1′-CH), 86.7 (4′-CH), 103.2 (5-CH), 127.0 (Ph-CH),128.4 (Ph-CH), 129.0 (Ph-CH), 139.8 (6-CH), 145.8 (Ph-C), 150.7 (2-C),163.7 (4-C).

ES⁺ m/z (%) 243 (Ph₃C⁺, 100), 470 ([M+H]⁺ , 4), 492 ([M+Na]⁺, 23).

HRMS (ES⁺) Found [M+H]⁺ 470.2076; C₂₈H₂₈N₃O₄ requires 470.2074.

M.p. 132-134° C.

Anal calcd for C₂₈H₂₇N₃O₄ (%): 0.53 HCl C 68.79; H, 5.68; N, 8.60 found:C, 68.79; H, 5.55; N, 8.59.

Example 15

3′-O-tertbutyldimethylsilyl-5′-Tritylamino-2′,5′-dideoxyuridine (15)

A solution of 5′-tritylamino-2′,5′-dideoxyuridine (0.172 g, 0.37 mmol)in anhydrous DMF (2 mL) was added drop-wise to an ice cold solution oftert-butyl dimethylsilyl chloride (68 mg, 0.45 mmol) and imidazole (60mg, 0.88 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirredat 0° C. for 2 h and at room temperature for a further 20 h. It was thenpartitioned between water (10 mL) and Et₂O (2×20 mL). The combinedorganic layers were washed with a saturated aqueous solution of NaHCO₃(15 mL), dried over MgSO₄ and concentrated in vacuo. The resultant whitesolid was further purified by column chromatography (Isolute SI column)using a gradient elution of 0→10% CH₃OH in CHCl₃. The fractions withR_(f)=0.69 (10% CH₃OH/CHCl₃) were pooled and evaporated to dryness toyield the title compound as a white solid (154 mg, 72%).

¹H NMR (300 MHz, CDCl₃) δ 0.00-0.02 (6H, 2×s, Si(CH₃)₂), 0.86 (9H, s,C(CH₃)₃), 1.90 (2H, m, 2′-H), 2.11-2.35 (2H, m, 5′-H), 2.59 (1H, bd,J≈13 Hz, 1″-NH), 4.06 (2H, m, 3′-H and 4′-H), 5.65 (1H, d, J=8.1 Hz,5-H), 6.25 (1H, t, J=6.3 Hz, 1′-H), 7.07 (1H, d, J=8.1 Hz, 6-H),7.14-7.37 (9H, m, Ph-H), 7.48 (6H, m,).

¹³C NMR (75 MHz, CDCl₃) δ-4.4 (SiCH₃), −4.2 (SiCH₃), 18.4 (C(CH₃)₃),26.1 (C(CH₃)₃), 41.4 (2′-CH₂), 46.5 (5′-CH₂), 71.2 (2″-C), 73.3 (3′-CH),85.6 (1′-CH), 87.3 (4′-CH), 103.1 (5-CH), 127.0 (Ph-CH), 128.4 (Ph-CH),129.0 (Ph-CH), 139.7 (6-CH), 145.9 (Ph-C), 150.6 (2-C), 163.7 (4-C).

ES⁺ m/z (%) 584 ([M+H]⁺), 606 ([M+Na]⁺).

HRMS (ES⁺) Found [M+H]⁺ 584.2938; C₃₄H₄₂N₃O₄Si requires 584.2939.

Example 16

1-(4-Fluoro-5-trityloxymethyl-tetrahydro-furan-2-yl)2,3-dihydro-1H-pyrimidin-4-oneor 3′-Fluoro-5′-O-trityl-2′,3′-dideoxyuridine (16)

3′-Fluoro-2′,3′-dideoxyuridine (0.3 g, 1.30 mmol) and triphenylmethylchloride (0.44 g, 1.57 mmol) were stirred in dry pyridine (20 ml)overnight at 50° C. under an atmosphere of nitrogen. The reactionmixture was then poured into ice-H₂O (50 ml) with vigorous stirring andfiltered. The precipitate was dissolved in EtOAc (50 ml) and thesolution was washed with 0.5M HCl (50 ml) and H₂O (50 ml) dried (Na₂SO₄)and reduced in vacuo to obtain a crude product, which was purified bygradient flash column chromatography eluting with 2→6% MeOH/CHCl₃ toobtain the title compound as a white solid (0.48 g, 77%).

¹H NMR (300 MHz; CDCl₃): δ 2.27-2.50 (1H, m, 2′-H), 2.78-2.92 (1H, m,2′-H), 3.53-3.63 (2H, m, 5′H), 4.41-4.51 (1H, d, J=27.3 Hz, 4′-H),5.33-5.53 (2H, m, 3′, 5-H), 6.50-6.55 (1H, m, 1′-H), 7.46 (15H, m,Ph-H), 7.80 (1H, d, J 8.1, 6-H);

¹³C NMR (75 MHz; CDCl₃): δ 39.43 and 39.71 (2′-CH₂), 63.75 and 63.89(5′-CH₂), 84.54 and 84.88 (4′-CH), 85.38 (1′-CH), 88.27 (Ph-C)—, 93.44(Ph-CH), 95.80 (Ph-CH), J 178.48, 3′-CH), 103.08 (5-CH), 128.06 (Ph-CH),128.58 (Ph-CH), 129.00 (Ph-CH), 140.18 (6-CH), 143.31 (Ph-C), 150.67(2-C), 163.53 (4-C);

¹⁹F NMR (282 MHz; CDCl₃): δ −174.26;

MS (Cl/NH₃., m/z): 473.2 (M+H⁺, 50%), 490.3 (M+NH₄ ⁺, 80%);

HRMS (EI., M⁺): Calculated for C₂₈H₂₅N₂O₄F, requires 472.1798, found472.1797.

IR_(vmax)/cm⁻¹ (KBr): 703 (s) and 763(s) (Aromatic, monosubstituted),1107.9 (C—F), 1689.3 (C═O) and 1702.3 (C═O). R_(f) (10% MeOH/CHCl₃):0.52.

Mp: 128-130° C.

Example 17

3′-Fluoro-5′-tritylamino-2′,3′,5′-trideoxyuridine (17)

The title compound was obtained as a light yellow crystalline solid (91mg, 32%) from the reaction of the corresponding amine (0.137 g, 0.59mmol) and trityl chloride (0.199 g, 0.66 mmol) in dry pyridine (4 mL).The procedure was similar to that followed for the preparation of the 3′hydroxy analogue 5′tritylamino-2′,5′-dideoxyuridine (WSP871, see example14).

¹H NMR (300 MHz, CDCl₃), δ 1.87-2.13 (2H, m, 2′-H), 2.28 (1H, dd, J=8.1,12.0 Hz, 1″-NH), 2.57-2.78 (2H, m, 5′-H), 4.48 (1H, dm, J≈25 Hz, 4′-H),5.11 (1H, dd, J=5.3, 53.7 Hz, 3′-H), 5.71 (1H, d, J=8.1 Hz, 5-H), 6.37(1H, dd, J=5.6, 8.7 Hz, 1′-H), 6.98 (1H, d, J=8.1 Hz, 6-H), 7.23-7.43(9H, m, Ph-H), 7.53 (6H, m, Ph-CH), 9.39 (1H, s, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 38.5 (d, J=21.8 Hz, 2′-CH₂), 46.1 (d, J=9.2Hz, 5′-CH₂), 71.1 (Ph₃C), 85.2 (d, J=25.3 Hz, 4′-CH), 85.5 (1′-CH), 94.4(d, J=179.9 Hz, 3′-CH), 103.6 (5-CH), 127.1 (Ph-CH), 128.5 (Ph-CH),128.9 (Ph-CH), 139.3 (6-CH), 145.7 (Ph-C), 150.5 (2-C), 163.4 (4-C).

¹⁹F NMR (282 MHz, CDCl₃) δ-175.7 (m, 3′-F).

ES⁺ m/z (%) 243 (Ph₃C⁺, 93), 494 ([M+Na]⁺, 92).

Example 18

3′-Fluoro-5′-O-triphenylsilyl-2′,3′-dideoxyuridine (18)

The title compound was synthesised following a similar procedure asdescribed for Example 11. 3′-Fluoro-2′,3′-dideoxyuridine (0.214 g, 0.93mmol) was reacted with triphenylsilyl chloride (0.332 g, 1.12 mmol) indry pyridine (7 mL) for 3 h. to yield the title compound as a whitesolid (0.274 g, 60%).

¹H NMR (300 MHz, CDCl₃) δ 2.19 (1H, m, 2′-H), 2.67 (1H, m, 2′-H), 4.11(2H, m, 5′-OH), 4.36 (1H, d, J=27.1 Hz, 3′-H), 5.22 (1.5H, m, 4′-H and5-H), 5.40 (0.5H, d, J=4.8 Hz, 4′-H), 6.50 (1H, dd, J=5.4, 9.1 Hz,1′-H), 7.41-7.75 (16H, m, 6-H and Ph-H), 9.04 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 39.5 (d, J=20.7 Hz, 2′-CH₂), 64.3 (d, J=11.5Hz, 5′-CH₂), 85.2 (1′-CH), 85.4 (d, J=24.7 Hz, 4′-CH), 94.9 (d, J=178.7Hz, 3′-CH), 103.1 (5-CH), 128.8 (Ph-CH), 131.2 (Ph-CH), 133.0 (Ph-C),135.7 (Ph-CH), 140.1 (6-CH), 150.7 (2-C), 163.5 (4-C).

¹⁹F NMR (282 MHz, CDCl₃) δ-175.1 (m, 3′-F).

ES⁺ m/z (%) 511 ([M+Na]⁺, 5), 87 (100).

ES⁻ m/z (%) 487 ([M−H⁺], 31), 75 (100).

Example 19

3′-Fluoro-5′-O-tert-Butyldiphenylsilyl-2′,3′-dideoxyuridine (19)

The title compound was synthesised following a similar procedure asdescribed for Example 12. 3′-Fluoro-2′,3′-dideoxyuridine (0.176 g, 0.77mmol) was reacted with tert-butyldiphenylsilyl chloride (0.238 g, 0.87mmol) and imidazole (0.116 g, 1.70 mmol) in dry DMF (4 mL) for 3 h.Compound WSP948 was obtained as a white solid (0.331 g, 92%).

¹H NMR (300 MHz, CDCl₃) δ 1.17 (9H, m, tBu-H), 2.24 (1H, m, 2′-H), 2.78(1H, m, 2′-H), 4.00 (2H, m, 5′-H), 4.38 (1H, d, J=26.7 Hz, 4′-H), 5.34(1H, dd, J=4.9, 53.8 Hz, 3′-H), 5.56 (1H, d, J=8.1 Hz, 5-H), 6.51 (1H,m, 1′-H), 7.43-7.60 (6H, m, Ph-H), 7.65-7.74 (4H, m, Ph-H), 7.27 (1H, d,J=8.1 Hz, 6-H), 9.11 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 19.7 (tBu-C), 27.4 (tBu-CH₃), 39.7 (d, J=21.3Hz, 2′-CH₂), 64.1 (d, J=10.9 Hz, 5′-CH₂), 85.4 (1′-CH), 85.6 (d, J=24.7Hz, 4′-CH), 94.7 (d, J=178.7 Hz, 3′-CH), 103.2 (5-CH), 128.5 (Ph-CH),128.6 (Ph-CH), 130.7 (Ph-CH), 132.2 (Ph-C), 132.8 (Ph-C), 135.7 (Ph-CH),136.0 (Ph-CH), 140.0 (6-CH), 150.6 (2-C), 163.5 (4-C).

¹⁹F NMR (282 MHz, CDCl₃) δ-175.5 (m, 3′-F).

ES⁻ m/z (%) 467 ([M−H⁺], 53), 75 (100).

Example 20

5′-O-paramethoxytrityl-2′-deoxyuridine (20)

4-Methoxytrityl (0.610 g, 1.98 mmol) was added to a solution of2′-deoxyuridine (0.410 g, 1.80 mmol) in anhydrous pyridine (10 mL). Thereaction mixture was stirred at 50° C. for 40 h. The crude mixture waspartitioned between water (40 mL) and DCM (2×40 mL). The organic layerswere combined, washed with water (2×80 mL), dried over MgSO₄ andconcentrated in vacuo. The resultant yellow oil was further purified bysilica gel column chromatography (using Jones Chromatography (solute SIcolumns). The column was eluted with a gradient of 0→5% CH₃OH in CHCl₃.The fractions with R_(f)=0.28 (10% CH₃OH/CHCl₃) yielded the titlecompound as a white crystalline solid (0.625 g, 69%).

¹H NMR (300 MHz, CDCl₃) δ 2.27 (1H, m, 5′-H), 2.42 (1H, m, 5′-H), 2.57(1H, bs, 3′-OH), 3.42 (2H, m, 2′-H), 3.76 (3H, s, OCH₃), 4.00 (1H, m,4′-H), 4.54 (1H, m, 3′-H), 5.37 (1H, d, J=8.1 Hz, 5-H), 6.29 (1H, t,J=6.3 Hz, 1′-H), 6.82 (2H, m, Ar—H), 7.18-7.38 (12H, m, Ar—H), 7.74 (1H,d, J=8.1 Hz, 6-H), 9.20 (1H, bs, 3—NH).

¹³C NMR (75 MHz, CDCl₃) δ 41.6 (2′-CH₂), 55.7 (OCH₃), 63.5 (5′-CH₂),71.9 (3′-CH), 85.5 (4′-CH), 86.5 (1′-CH), 87.8 (Ar₃C), 102.7 (5-CH),113.8 (Ar—CH), 127.7 (Ar—CH), 128.5 (Ar—CH), 128.8 (Ar—CH), 130.8(Ar—CH), 135.1 (Ar—C), 140.6 (6-CH), 144.1 (Ar—C), 144.3 (Ar—C), 150.8(2-C), 159.3 (Ar—C), 163.7 (4-C).

ES⁺ m/z (%) 523 ([M+Na]⁺, 100)

HRMS (ES⁺) Found [M+Na]⁺523.1848; C₂₉H₂₈N₂O₆Na requires 523.1845.

IR (KBr) 3208, 3054, 1714, 1694, 1682, 1507, 1470, 1250, 1092, 1035, 759cm⁻¹.

M.p. 96-97° C.

Anal calcd for C₂₉H₂₈N₂O₆ (%): 1.43 HCl, 0.40 H₂O C, 62.21; H, 5.44; N,5.00; Cl, 9.06; found: C, 62.17; H, 5.05; N, 4.85, Cl, 8.86.

Example 21

5′-O-(4-cyanotrityl)-2′-deoxyuridine (21)

4-Cyanotrityl (0.397 g, 1.31 mmol) was added to a solution of2′-deoxyuridine (0.229 g, 1.00 mmol) in dry pyridine (5 mL). As thereaction was not complete after 72 h at 50° C., DMAP (11 mg, 0.09 mmol)was added and the reaction mixture was kept at 50° C. for a further 20h. H₂O (20 mL) was added and the crude mixture was extracted with DCM(2×15 mL and 10 mL). The combined organic layers were dried over Na₂SO₄,concentrated in vacuo and purified by flash column chromatographyeluting the column (ISOLUTE SI) with a gradient of 0→6% CH₃OH in CHCl₃.The fractions with R_(f)=0.29 (10% CH₃OH/CHCl₃) afforded the titlecompound as a white solid (0.215 g, 43%).

¹H NMR (300 MHz, CDCl₃) δ 2.19 (1H, m, 2′-CHH), 2.45 (1H, m, 2′-CHH),2.94 (1H, bs, 3′-OH), 3.38 (2H, m, 5′-H), 4.06 (1H, m, 4′-H), 4.50 (1H,m, 3′-H), 5.45 (1H, d, J=8.1 Hz, 5-H), 6.27 (1H, t, J=6.2 Hz, 1′-H),7.24-7.34 (10H, m, Ph-H), 7.53-7.60 (5H, m, 6-H and Ar—H), 9.50 (1H, bs,3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 41.4 (2′-CH₂), 63.9 (5′-CH₂), 71.7 (3′-CH),85.6 (1′-CH), 86.1 (4′-CH), 87.6 (ArPh₂ C), 102.8 (5-CH), 111.4 (Ar—C),119.0 (C≡N), 128.6 (Ph-CH), 128.8 (Ph-CH), 129.0 (Ar—CH), 132.4 (Ar—CH),140.3 (6-CH), 142.0 (Ph-C), 142.1 (Ph-C), 150.1 (Ar—C), 150.8 (2-C),163.8 (4-C).

ES⁺ m/z (%) 518 ([M+Na]⁺, 23), 268 (CNTr⁺, 100).

ES⁻ m/z (%) 494 (M−H⁺, 100).

HRMS (ES⁺) Found [M+NH₄]⁺513.2132; C₂₉H₂₉N₄O₅ ⁺ requires 513.2132.

M.p. 92-95° C.

IR (KBr) 3401, 3180, 3060, 2230 (CN), 1685, 1463, 1273, 1088 cm⁻¹.

Anal calcd for C₂₉H₂₅N₃O₅ (%): 2.35 HCl C, 59.93; H, 4.74; N, 7.23found: C, 59.89; H, 4.45; N, 7.02.

Example 22

5′-[(4-cyanotrityl)amino]-2′,5′-dideoxyuridine (22)

4-Cyanotrityl chloride (0.406 g, 1.34 mmol) was added to a solution of5′-amino-2′,5′-dideoxyuridine (0.239 g, 1.05 mmol) in dry pyridine (5mL). The reaction mixture was stirred at 40° C. for 48 h. The reactionmixture was filtered and the filtrate concentrated in vacuo.Purification was carried out using flash column chromatography elutingthe column (ISOLUTE SI) with a gradient of 0→5% CH₃OH in CHCl₃. Thefractions with R_(f)=0.31 (10% CH₃OH/CHCl₃) afforded the title compoundas a white crystalline solid (0.386 g, 37%).

¹H NMR (300 MHz, CDCl₃) δ 2.03 (2H, m, 2′-CHH and 5′-NH), 2.21 (1H, m,5′-CHH), 2.37 (1H, m, 2′-CHH), 2.58 (1H, m, 5′-CHH), 3.31 (1H, bs,3′-OH), 4.10 (1H, m, 4′-H), 4.24 (1H, m, 3′-H), 5.63 (1H, d, J=8.1 Hz,5-H), 6.24 (1H, t, J=6.3 Hz, 1′-H), 7.00 (1H, dd, J=2.1, 8.1 Hz, 6-H),7.18-7.48 (10H, m, Ph-H), 7.54 (2H, d, J=8.1 Hz, Ar—H), 7.66 (2H, d,J=8.1 Hz, Ar—H), 9.74 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 40.7 (2′-CH₂), 46.5 (5′-CH₂), 71.3 (ArPh₂C),72.8 (3′-CH), 85.6 (1′-CH), 86.5 (4′-CH), 103.3 (5-CH), 110.7 (Ar—CH),119.2 (CN), 127.6 (Ph-CH), 128.7 (Ph-CH), 128.9 (Ph-CH), 129.1 (Ph-CH),129.5 (Ar—CH), 132.4 (Ar—CH), 139.9 (6-CH), 144.3 (Ph-C), 145.0 (Ph-C),150.8 (2-C), 151.6 (Ar—C), 163.8 (4-C).

ES⁺ m/z (%) 517 ([M+Na]⁺, 19), 495 ([M+H]⁺ , 9), 517 (CNTr⁺, 100).

ES⁻ m/z (%) 493 (M−H⁺, 19), 111 (uracil-W, 100).

HRMS (ES⁺) Found [M+H]⁺ 495.2023; C₂₉H₂₇N₄O₄ ⁺ requires 495.2027.

M.p. 160-163° C.

IR (KBr) 3387, 3177, 3027, 2230 (CN), 1699, 1661, 1466, 1267, 1097, 1039cm⁻¹.

Anal calcd for C₂₉H₂₉N₃O₅, 0.98 (%): HCl, 0.40H₂O C, 64.80; H, 5.21; N,10.42; Cl, 6.46; found: C, 64.69; H, 4.90; N, 10.28; Cl, 6.84.

Example 23

5′-paraMethoxytritylamino-2′,5′-dideoxyuridine (23)

The procedure was similar to that described for example 20.5′-Amino-2′,5′-dideoxyuridine (0.204 g, 0.90 mmol) was reacted with4-methoxytrityl (0.292 g, 0.99 mmol) to yield the title compound as awhite solid (0.115 g, 26%).

¹H NMR (300 MHz, CDCl₃) δ 2.04 (2H, m, 2′-H), 2.29-2.48 (2H, m, 5′-H),2.69 (1H, dd, J=3.7, 12.1 Hz, 5′—NH), 3.83 (3H, s, OCH₃), 4.15 (1H, m,4′-H), 4.30 (1H, m, 3′-H), 5.69 (1H, d, J=8.1 Hz, 5-H), 6.32 (1H, t,J=6.4 Hz, 1′-H), 6.88 (2H, m, Ar—H), 7.17 (1H, d, J=8.1 Hz, 6-H),7.21-7.61 (12H, m, Ar—H).

¹³C NMR (75 MHz, CDCl₃) δ 40.8 (2′-CH₂), 46.6 (5′-CH₂), 55.7 (OCH₃),70.7 (Ar₃C), 73.0 (3′-CH), 85.4 (4′-CH), 86.8 (1′-CH), 103.2 (5-CH),113.7 (Ar—CH), 126.9 (Ar—CH), 128.4 (Ar—CH), 128.9 (Ar—CH), 130.2(Ar—CH), 138.0 (Ar—C), 139.9 (6-CH), 146.2 (Ar—C), 150.8 (2-C), 158.4(Ar—C), 163.8 (4-C).

ES⁺ m/z (%) 522 ([M+Na]⁺, 27).

HRMS (ES⁺) Found [M+H]⁺ 500.2174; C₂₉H₃₀N₃O₅ ⁺ requires 500.2180.

IR (KBr) 3052, 1713, 1694, 1682, 1666, 1650, 1506, 1250, 1034, 760 cm⁻¹.

M.p. 140-142° C.

TLC (10% CH₃OH/CHCl₃) R_(f)=0.29.

Anal (%) found C, 65.69; H, 5.52; N, 7.86, Cl, 6.05;

Calcd for C₂₉H₂₉N₃O₅, 0.87 HCl C, 65.56; H, 5.67; N, 7.91, Cl 5.81.

Example 24

5′-[(2-chlorotrityl)amino]-2′,5′-dideoxyuridine (24)

5′-Amino-2′,5′-dideoxyuridine (0.237 g, 1.04 mmol) was reacted with2-chlorotrityl chloride (0.415 g, 1.33 mmol) in dry pyridine (5 mL) at40° C. for 24 h. A second portion of 2-chlorotrityl chloride (0.198 g,0.63 mmol) was added. After a further 2 h stirring at 40° C., thereaction was quenched with MeOH (2 mL). The reaction mixture wasconcentrated in vacuo and purified by flash column chromatography usingan ISOLUTE SI column eluted with a gradient of 0→5% CH₃OH in CHCl₃. Thefractions with R_(f)=0.17 (10% CH₃OH/CHCl₃) yielded the title compoundas a white solid (85 mg, 16%).

¹H NMR (300 MHz, CDCl₃) δ 2.02 (1H, m, 2′-H), 2.20 (1H, m, 5′-H), 2.37(1H, m, 2′-H), 2.53 (1H, m, 5′-H), 4.16 (1H, m, 3′-H), 4.24 (1H, m,4′-H), 5.62 (1H, d, J=8.1 Hz, 5-H), 6.26 (1H, t, J=6.4 Hz, 1′-H),7.05-7.45 (15H, m, 6-H and Ar—H).

¹³C NMR (75 MHz, CDCl₃) δ 41.1 (2′-CH₂), 47.3 (5′-CH₂), 71.5 (ArPh₂C),72.9 (3′-CH), 85.5 (1′-CH), 86.8 (4′-CH), 103.0 (5-CH), 126.8 (CITr-CH),126.9 (CITr-CH), 127.6 (CITr-CH), 128.4 (CITr-CH), 128.5 (CITr-CH),128.6 (CITr-CH), 129.2 (CITr-CH), 132.3 (CITr-CH), 132.9 (CITr-CH),134.7 (CITr-C), 140.0 (6-CH), 140.9 (CITr-C), 144.8 (CITr-C), 146.0(CITr-C), 150.7 (2-C), 163.7 (4-C).

ES⁺ m/z (%) 526 ([M+Na]⁺, 11), 504 ([M+H]⁺ , 14), 277 (CITr⁺, 100).

ES⁻ m/z (%) 504 (M−H⁺, 11), 111 (uracil-W, 100).

HRMS (ES⁺) Found [M+H]⁺ 504.1689; C₂₈H₂₇N₃O₄Cl⁺ requires 504.1685.

M.p. 129-131° C.

Example 25

5′-Triphenylsilyloxy-2′,3′-dideoxydidehydrouridine (25)

To a solution of 2′,3′-dideoxydidehydrouridine (0.316 g, 1.50 mmol) indry pyridine (5 mL) cooled in an ice-salt bath was added drop-wise asolution of triphenylsilyl chloride (0.595 g, 2.02 mmol) in dry pyridine(3 mL). The reaction mixture was kept at 0° C. under nitrogen for 2 h30.As TLC monitoring evidenced the presence of unreacted starting material,additional triphenylsilyl chloride (0.296 g, 1.00 mmol) in dry pyridine(1 mL) was added. After 1 h30 min the reaction was quenched with CH₃OH(50 uL). Removal of the solvent in vacuo afforded a crude white gumwhich was purified by silica gel chromatography (Jones Chromatography(solute SI column) eluted with 0→5% CH₃OH in CHCl₃. The title wasobtained as a white solid (0.476 g, 68%) from the fractions withR_(f)=0.57 (10% CH₃OH/CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 4.04 (1H, dd, J=2.2, 11.7 Hz, 5′-H), 4.19 (1H,dd, J=2.5, 11.7 Hz, 5′-H), 4.78 (1H, dd, J=1.9, 8.1 Hz, 5-H), 4.98 (1H,m, 4′-H), 5.90 (1H, d, J=5.7 Hz, 1′-H), 6.33 (1H, dd, J=1.4, 4.5 Hz,2′-H), 7.12 (1H, m, 3′-H), 7.40-7.65 (15H, m, Ph-H), 7.80 (1H, d, J=8.1Hz, 6-H), 8.99 (1H, bs, 3-NH).

¹³C NMR (75 MHz, CDCl₃) δ 64.7 (5′-CH₂), 87.4 (1′-CH), 90.0 (4′-CH),102.6 (5-CH), 127.1 (2′-CH), 128.6 (Ph-CH), 131.0 (Ph-CH), 133.3 (Ph-C),134.9 (3′-CH), 135.8 (Ph-CH), 141.5 (6-CH), 151.2 (2-C), 163.6 (4-C).

ES⁺ m/z (%) 491 ([M+Na]⁺, 36), 119 (100).

HRMS (ES⁺) Found [M+NH₄]⁺446.1887; C₂₅H₂₈N₃OSi⁺ requires 446.1894.

M.p. 73-74° C.

Anal (%) found C, 67.75; H, 5.04; N, 5.84; Cl, 1.89;

Calcd for C₂₇H₂₇N₂O₄Si, 0.25 HCl C, 67.89; H, 5.12; N, 5.86; Cl, 1.86.

Example 26

5′-Pixylamino-2′,5′-dideoxyuridine (26)

2′-Amino-2′,5′-deoxyuridine (0.231 g, 1.02 mmol) was reacted with pixylchloride (0.390 g, 1.33 mmol) in dry pyridine (5 mL) at 40° C. for 48 h.H₂O (10 mL) was added and the crude mixture was extracted with DCM (2×15mL). The organic layers were dried over Na₂SO₄, concentrated in vacuoand purified by flash column chromatography eluting the column (ISOLUTESI) with a gradient of 0→10% CH₃OH in CHCl₃. The fractions withR_(f)=0.11 (10% CH₃OH/CHCl₃) yielded the title compound as a white solid(0.118 g, 24%).

¹H NMR (300 MHz, CDCl₃) δ 1.31 (1H, m, 2′-CHH), 2.04 (1H, m, 2′-CHH),2.29 (1H, dd, J=4.3, 13.8 Hz, 5′-CHH), 2.58 (1H, m, 5′-CHH), 3.84 (2H,m, 3′-H and 4′-H), 5.69 (1H, d, J=8.1 Hz, 5-H), 6.21 (1H, t, J=6.5 Hz,1′-H), 7.03-7.53 (14H, m, 6-H and Ar—H).

¹³C NMR (75 MHz, CDCl₃) δ 39.4 (2′-CH₂), 42.3 (5′-CH₂), 72.1 (3′-CH),76.9 (pixyl-C), 84.7 (1′-CH), 86.1 (4′-CH), 103.1 (5-CH), 116.8(pixyl-CH), 116.9 (pixyl-CH), 123.3 (pixyl-C), 123.6 (pixyl-C), 123.97(pixyl-CH), 124.03 (pixyl-CH), 127.4 (pixyl-CH), 127.6 (—CH), 128.2(pixyl-CH), 130.20 (pixyl-CH), 130.24 (pixyl-CH), 131.1 (pixyl-CH),131.5 (pixyl-CH), 140.3 (6-CH), 148.0 (pixyl-C), 150.5 (2-C), 151.4(pixyl-C), 151.9 (pixyl-C), 163.5 (4-C).

ES⁺ m/z (%) 506 ([M+Na]⁺, 4), 257 (Pixyl⁺ that is C₁₉H₁₃O⁺, 100).

ES⁻ m/z (%) 482 (M−H⁺, 100).

HRMS (ES⁺) Found [M+H]⁺ 484.1871; C₂₈H₂₆N₃O₅ ⁺ requires 484.1867.

M.p. 117-119° C.

Anal calcd for C₂₈H₂₅N₃O₅ (%): 1.58 HCl C, 62.15; H, 4.95; N, 7.7; 7found C, 62.07; H, 4.66; N, 7.50.

Example 27

5′-O-trityl-2′-deoxyuridine (27)

2″-deoxyuridine (4.00 g, 17.5 mmol) and triphenylmethyl chloride (5.37g, 19.25 mmol) were stirred in anhydrous pyridine (70 ml) at 50° C.overnight. Additional triphenylmethyl chloride (1.00 g, 3.59 mmol) wasadded after 18 hours, and the mixture was stirred for a further 4 hoursat 50° C. The reaction mixture was then poured into ice-H₂O (300 ml) andstirred vigorously. The precipitate was extracted with EtOAc (3×100 ml).The organic solution was then washed with 0.5 M HCl (4×100 ml), driedwith MgSO₄ and filtered. The filtrate was then washed further withEtOAc, which was then evaporated, and finally with DCM. Solvent wasremoved using a Buchi rotary evaporator, and finally with the vacuumpump.

White solid (7.69 g, 93%).

Example 28

3′-O-Mesyl-5″-O-trityl-2″-deoxyuridine (28)

Methanesulphonyl chloride (0.173 ml, 2.24 mmol) was added to a solutionof (8) (0.30 g, 0.64 mmol) in anhydrous pyridine (5 ml) with ice-bathcooling. The mixture was stirred for 4 hours at room temperature. Afterthis time, ice-water (1 ml) was added; the mixture was stirred for 5minutes, then poured into ice-water (30 ml) and filtered. Theprecipitate was dissolved in CHCl₃ (30 ml), the solution was washed with0.5 M HCl (10 ml) and water (3×10 ml), dried (MgSO₄), filtered, andreduced in vacuo which gave the title product as a yellow/orange solid(0.29 g, 83%).

Example 29

2,3′-Anhydro-5′-O-trityl-2′,3′-dideoxyuridine (29)

DBU (1.00 ml, 7.12 mmol) and compound 28 (3.55 g, 6.48 mmol) werestirred in DCM (25 ml) over 30 hours. The mixture was washed with water(2×30 ml), the organic layer was dried (MgSO₄), filtered and reduced invacuo. The residue was purified by column chromatography (5% MeOH/CHCl₃)which gave the title product as a white solid (2.42 g, 82%).

Example 30

3′-Azido-5″-O-trityl-2′,3′-dideoxyuridine (30)

Lithium fluoride (0.145 g, 5.61 mmol) was suspended in DMF (3 ml) andheated to 105° C. with stirring. To the stirred suspension was addedN,N,N′,N′-tetramethylethylenediamine (5 ml) followed byazidotrimethylsilane (0.64 g, 5.61 mmol). After stirring for an hour,compound 29 (1.41 g, 3.11 mmol) dissolved in N,N-dimethylformamide (2ml) was added, and the reaction was allowed to proceed for 20 hours at110° C. The mixture was cooled, poured into CHCl₃ (110 ml) and filteredthrough Celite. The solvent was removed under reduced pressure and theresidue (brown oil) was taken in EtOAc (100 ml). The organic phase waswashed with water (4×180 ml), dried (MgSO₄), filtered and concentrated.The concentrated mixture was purified by column chromatography (3%MeOH/CHCl₃) which gave the title product as an orange solid (0.996 g,65%).

Example 31

3′-Amino-5′-O-trityl-2′,3′-dideoxyuridine (31)

Lindlar's catalyst (20 mg) was added to compound 30 (0.10 g, 0.20 mmol),and was then suspended in ethanol (5 ml). Air was removed from the flaskby flushing with nitrogen several times. The nitrogen was then removedand replaced with hydrogen. The mixture was stirred for 5 hours, andthen filtered through Celite. Fresh Lindlar's catalyst (20 mg) was addedto the filtrate. The flask was flushed with nitrogen and then hydrogenas previously, and the reaction was left stirring for another 3 hours.The reaction mixture was filtered through Celite. The solvent wasevaporated and the concentrated solution was purified by columnchromatography (MeOH/DCM 2%→10%) which gave the title product as a whitesolid (0.065 g, 70%). R_(f): 0.3 in DCM/MeOH 90:10.

¹H-NMR (300 MHz, CDCl₃): δ 7.97 (d, J=8.2 Hz, 1H, H-6), 7.47-7.27 (m,15H, H-aromatic), 6.20 (q, J=3.3 Hz, 1H, H-1′) 5.41 (d, J=8.1 Hz, 1H,H-5), 3.73-3.38 (m, 5H, H-3′, H-4′, H-5′), 2.44±2.18 (m, 2H, H-2′).

¹³C-NMR (75 MHz, CDCl₃): δ 163.5 (C-4), 150.5 (C-2), 143.7 (C-7′), 140.7(C-6), 129.1 (C-8′), 128.5 (C-9′), 127.8 (C-10′), 102.2 (C-5), 87.9(C-6′), 87.2 (C-4′), 85.2 (C-1′), 62.2 (C-5′), 50.7 (C-3′), 42.7 (C-2′).

LRMS: (ES+mode): m/z=491.7 [(M+Na)⁺, 45%]; m/z=243.2 [(Tr)⁺, 100%].

HRMS: (ES+mode): found 492.1902; required 492.1899 for C₂₈H₂₇N₃O₄Na [MNa]⁺.

Microanalysis calculated for C₂₈H₂₇N₃O₄×0.5H₂O:

C, 70.28; H, 5.90; N, 8.78%; found: C, 70.64; H, 5.92; N, 8.41%.

Example 32

3′-Acetylamino-5′-O-trityl-2′,3′-dideoxyuridine (32)

Compound 31 (0.10 g, 0.213 mmol) was suspended in DCM (5 ml), and tothis was added acetic anhydride (0.047 g, 0.044 ml, 0.469 mmol) andtriethylamine (0.065 ml, 0.469 mmol). The mixture was stirred at roomtemperature for 3 hours. After this time the solvent was evaporated togive a white solid. The product was purified by column chromatography(MeOH/DCM 2%→6%), and evaporation of the solvent gave the title compoundas a white solid (0.103 g, 95%).

R_(f): 0.45 in DCM/MeOH 90:10.

¹H-NMR (300 MHz, CDCl₃): δ 9.82 (s, 1H, N—H), 7.83 (d, J=8.2 Hz, 1H,H-6), 7.46-7.28 (m, 16H, H-aromatic) 6.94 (s, 1H, N—H), 6.34 (t, J=6.3Hz, 1H, H-1′), 5.39 (d, J=8.1 Hz, 1H, H-5), 4.79-4.72 (m, 1H, H-3′),4.07 (s, 1H, H-4′) 3.59-3.47 (m, 2H, H-5′), 2.50-2.32 (m, 2H, H-2′),2.04 (s, 3H, H-12′)

¹³C-NMR (75 MHz, CDCl₃): δ 163.6 (C-4), 154.9 (C-2), 143.6 (C-7′), 140.5(C-6), 129.1 (C-8′), 128.5 (C-9′), 127.9 (C-10′), 103.1 (C-5), 88.1(C-6′), 87.3 (C-1′), 85.2 (CH, C-4′), 62.1 (C-5′), 50.9 (C-3′), 38.8(C-2′)

LRMS: (ES+mode): m/z=533.8 [(M+Na)⁺, 20%].

HRMS: (ES+mode): Found 534.2009; required 534.2005 for C₃₀H₂₉N₃O₅Na[M+Na]³⁰

Microanalysis calculated for C₃₀H₂₉N₃O₅×1.0 HCl×1.0 H₂O

C, 63.66; H, 5.70; N, 7.42%; found C, 63.20; H, 5.15; N, 7.11%.

Example 33

Diphenyl(pyridin-2-yl)methanol (33)

A solution of 2-bromopyridine (5 g, 0.032 mol) in dry THF (15 0 mL) wascooled to −70° C. To this cooled solution was added n-BuLi (2.8 M, 12.4mL, 0.034 mol) over a period of 20 min and allowed to stir for 2 h underN₂ atmosphere. A solution of benzophenone (5.8 g, 0.032 mol) in dry THF(50 mL) was added to the solution at the same temperature over a periodof 30 min. The reaction mixture was warmed slowly to RT and allowed tostir another 5 h at RT. The reaction mixture was concentrated undervacuum and the residue was washed with petroleum ether. The organiclayer was filtered and the filtrate was concentrated under vacuum togive the title compound (8 g, 95%).

Example 34

2-[Chloro (diphenyl)methyl]pyridine hydrochloride (34)

To a mixture of diphenyl(pyridin-2-yl)methanol (4 g, 0.015 mol) inthionylchloride (50 mL) was added acetylchloride (15 mL, 0.195 mol) atRT and heated to 50° C. for 48 h. The reaction mixture was concentratedunder vacuum and the residue was azeotroped with dry benzene (100 mL×2)to give the title compound as the hydrochloride salt (4.4 g, >95%).

Example 35

4-[Hydroxy(diphenyl)methyl]benzonitrile (35)

The procedure described in example 49 was followed but using4-bromobenzonitrile (5 g, 0.027 mol) instead of 2-bromopyridine whichgave the title compound (7.5 g, 94%).

Example 36

4-[Chloro(diphenyl)methyl]benzonitrile (36)

To a mixture of 4-[hydroxy(diphenyl)methyl] in dry toluene (60 mL) wasadded acetylchloride (3 mL) at RT and heated to 50° C. for 12 h. Thereaction mixture was concentrated under vacuum. The residue wasrecrystallized from pet. ether to give the product (1.7 g, 40%).

Example 37

Diphenyl (pyrimidin-5-yl)methanol (37)

A solution of 5-bromopyrimidine (10 g, 0.063 mol) in a mixture of dryTHF (150 mL) and hexane (50 mL) was cooled to −100° C. To this cooledsolution was added n-BuLi (4 g, 21 mL, 0.062 mol) over a period of 30min and stirred for another 30 min. A solution of benzophenone (11.5 g,0.063 mol) in dry THF (50 mL) was added to this at the same temperatureover a period of 30 min. The reaction mixture was warmed slowly to RTand allowed to stir another 1 h at RT. The reaction was quenched withcold water (200 mL), ethyl acetate was added and the organic layer wasseparated. The organic layer was dried, concentrated and the crudeproduct was purified by column chromatography on silica gel (up-to 25%ethyl acetate in pet. ether) to give the product (8 g). TLC: Pet.ether/EtOAc, 1:1, R_(f)=0.3

Example 38

2,2,2-Triphenylethanol (38)

To a suspension of LAH (3.9 g, 0.104 mol) in dry THF (200 mL) wasstirred at 0° C. for 20 min. A solution of 2,2,2-triphenylacetic acid(10 g, 0.034 mol) in dry THF (50 mL) was added in a drop-wise manner.The reaction mixture was stirred at RT overnight. Excess LAH wasquenched with 1.5 N HCl and the reaction mixture was further stirred for2 h at RT. The reaction mixture was filtered through celite, washed withethyl acetate and the filtrate was concentrated under vacuum. The crudeproduct was purified by column chromatography on silica gel (4% ethylacetate in pet. ether) to give the title compound (4.6 g, 48%). TLC:Pet. ether/EtOAc, 7:3, R_(f)=0.2

Example 39

3,3,3-Triphenylpropan-1-ol (39)

To a magnetically stirred suspension of LAH (8.3 g, 0.219 mol) in dryTHF (50 mL) was added a solution of 3,3,3-triphenylpropionic acid (9.5g, 0.0314 mol) over a period of 30 min at 0° C. The reaction mixture wasallowed to stir at RT for 14 h. The reaction mixture was cooled andexcess LAH was quenched with 20% NaOH solution (50 mL). The reactionmixture was passed through celite, washed with THF and the filtrate wasconcentrated under vacuum. The residue was washed with pet. ether anddried which gave the title compound (8 g, >85%).

TLC: Pet. ether/EtOAc, 7:3, R_(f)=0.2

Example 40

1,1,2-Triphenylethanol (40)

To a suspension of Mg (1.7 g, 0.07 mol) in dry ether (25 mL) was added asolution of benzyl bromide (10 mL, 1.5 equ.) in dry ether (25 mL)drop-wise and allowed to stir at RT for 1 h. By the time all magnesiumwas dissolved and the reaction mixture was cooled to 0° C. To this wasadded a solution of benzophenone (10 g, 0.05 mol) in dry ether (25 mL)and allowed to stir at RT for 5 h. The progress of the reaction wasfollowed by TLC and when it was ready the reaction mixture was quenchedwith saturated NH₄Cl solution, extracted with ether (100 mL), washedwith brine, dried and concentrated under vacuum. The crudeproduct waspurified by column chromatography on silica gel (10% ethyl acetate inpet. ether) to give the title compound (9.6 g, 65%) as a white solid.

TLC: Pet. ether/EtOAc, 9:1, R_(f)=0.4

Example 41

4-Trityloxy-but-2-en-1-ol (41)

Trityl chloride (557 mg; 2 mmol) Et₃N (0.306 ml; 2.2 mmol) and DMAP (10mg; 0.08 mmol) were added to a emulsion of cis-2-buten-1,4-diol (1.76 g;20 mmol) in DCM (10 ml). The mixture was stirred at room temperatureunder atmosphere of nitrogen for 24 hours. After such period of time thecomplete disappearance of trityl chloride was observed by TLC(EtOAc/Hexane 50:50). DCM (20 ml) and water (10 ml) were added to themixture. The phases were separated and the organic layer was washed withwater (10 ml) and brine (10 ml). The solvent was dried over MgSO₄ andevaporated under reduced pressure affording a residue (white oil) whichwas purified by flash chromatography using Hexane/EtOAc 70:30→40:60 asgradient which gave the title product as a colourless oil (563 mg, 81%).

Example 42

Trans-2-buten-1,4-diol (42)

2-Butyn-1,4-diol (1 g; 11.64 mmol) was dissolved in dry THF (25 ml)under atmosphere of nitrogen. The solution was cooled to −78° C. with adry-ice/acetone bath. A cold solution of LAH in THF 1M (12.7 ml; 12.7mmol) was added with a syringe. The reaction was left worm to roomtemperature in 4 hours. The disappearance of the starting alkyne wasobserved by TLC (Hexane/EtOAc 30:70); then the solution was cooled to−0° C. with an ice bath and the quenched with NaOH 1M, until no gas wasdeveloped. The pH was adjusted to 8 with HCl 1M and then silica wasadded to the solution. The solvents were removed under reduced pressureand the residue was loaded into a chromatographic column and purifiedusing Hexane/EtOAc 30:70 as eluent which gave the title compound as acolourless oil (817 mg, 79%). R_(f): 0.11 in Hexane/EtOAc 30:70 (PMA)

¹H-NMR (300 MHz, CD₃OD): δ 5.83 (bs; 2H; H-2+H-3); 4.07 (d; J=3.57 Hz;4H; H-1+H-4)

¹³C-NMR (75 MHz, CD₃OD): δ 131.7 (C-2 & C-3); 63.4 (C-1 & C-4)

LRMS (ES+): m/z 111.0 [M+Na]⁺100%.

Example 43

(E)-4-(Trityloxy)but-2-en-1-ol (43)

A solution of Trityl chloride (500 mg; 1.81 mmol), TEA (0.277 ml; 1.99mmol) and DMAP (8.8 mg; 0.072 mmol) in dry DCM (5 ml) was added with asyringe to a solution of the diol (10) (800 mg; 9.07 mmol) in DCM (15ml). The mixture was stirred at room temperature for 1 hour and 30minutes, then other TrCl, TEA and DMAP (half quantities than before)were added. The reaction was stirred at the same temperature until TLC(Hexane/EtOAc 50:50) showed complete disappearance of Trityl chloride.After 1.5 hours water (20 ml) was added and the reaction was stirred forfew minutes, then the phases were separated. The organic layer waswashed with water (25 ml) and brine (25 ml). The solvent was dried overMgSO₄ and evaporated to afford a crude oil which was purified by flashchromatography using Hexane/EtOAc 50:50 as eluent which gave the titlecompound as a colourless oil, 637 mg, 71%. R_(f): 0.72 in Hexane/EtOAc50:50 (UV/PMA).

¹H-NMR (300 MHz, CDCl₃): δ 7.54-7.51 (m; 6H; H-7); 7.39-7.27 (m; 9H;H-8+H-9); 6.12-6.03 (m; 1H; H-3); 5.91-5.83 (m; 1H; H-2); 4.24 (bs; 2H;H-4); 3.71-3.71 (m; 2H; H-1).

¹³C-NMR (75 MHz, CDCl₃): δ 144.6 (C-6); 130.5 (C-3 & C-2); 129.0 (C-8);128.7 (C-7); 127.4 (C-9); 87.3 (C-5); 64.5 (C-4); 63.8 (C-1).

LRMS (ES+): m/z 331.2 [M+H]⁺ 100%.

Example 44

5-Trityloxypentanol (44)

The procedure described in example 41 was followed but using1,5-pentanediol (376 mg, 3.6 mmol) as alcohol instead ofcis-2-buten-1,4-diol in the reaction with trityl chloride, which gavethe title compound (300 mg, 24%).

Example 45

5-(Tritylamino)-pentan-1-ol (45)

The title compound (24%) was prepared as described in example 41 butusing 5-aminopentanol instead of cis-2-buten-1,4-diol in the reactionwith trityl chloride.

Biological Examples Example B1 Malaria Whole Cell Assays ParasiteCultures

Two strains of P. falciparum are used in this study: The drug sensitiveNF54 (an airport strain of unknown origin) and K1 (Thailand, chloroquineand pyrimethamine resistant). The strains are maintained in RPMI-1640medium with 0.36 mM hypoxanthine supplemented with 25 mM HEPES, 25 mMNaHCO₃, neomycin (100 U/ml) and Albumax® (lipid-rich bovine serumalbumin) (GIBCO, Grand Island, N.Y.) (5 g/l), together with 5% washedhuman A+ erythrocytes. All cultures and assays are conducted at 37° C.under an atmosphere of 4% CO₂, 3% O₂ and 93% N₂. Cultures are kept inincubation chambers filled with the gas mixture. Subcultures are dilutedto a parasitaemia of 0.1-0.5% and the medium changed daily.

Drug Sensitivity Assays

Antimalarial activity is assessed using an adaptation of the proceduresdescribed by Desjardins et al. (Antimicrob. Agents Chemother.16(6):710-8, 1979), and Matile and Pink (In: Lefkovits, I. and Pernis,B. (Eds.). Immunological Methods. Academic Press, San Diego, pp.221-234, 1990).Stock drug solutions are prepared in 100% DMSO (dimethylsulfoxide) at 10mg/ml, unless otherwise suggested by the supplier, and heated orsonicated if necessary. After use the stocks are kept at −20° C. Thecompound is further diluted to the appropriate concentration usingcomplete medium without hypoxanthine.Assays are performed in sterile 96-well microtiter plates, each wellcontaining 200 μl of parasite culture (0.15% parasitemia, 2.5%hematocrit) with or without serial drug solutions. Seven 2-folddilutions are used covering a range from 5 μg/ml to 0.078 μg/ml. Foractive compounds the highest concentration is lowered (e.g. to 100ng/ml), for plant extracts the highest concentration is increased to 50μg/ml. Each drug is tested in duplicate and repeated once for activecompounds showing an IC₅₀ below 0.5 μg/ml. After 48 hours of incubationat 37° C., 0.5 μCi ³H-hypoxanthine is added to each well. Cultures areincubated for a further 24 h before they are harvested onto glass-fiberfilters and washed with distilled water. The radioactivity is countedusing a Betaplate™ liquid scintillation counter (Wallac, Zurich,Switzerland). The results are recorded as counts per minute (CPM) perwell at each drug concentration and expressed as percentage of theuntreated controls. From the sigmoidal inhibition curves IC₅₀ values arecalculated.

Primary Screen

K1 strain is used. The compounds are tested at 7 concentrations (5000 to78 ng/ml).Artemisinin and chloroquine are included as reference drugs.If the IC₅₀ is >5 μg/ml, the compound is classified as inactiveIf the IC₅₀ is 0.5-5 μg/ml, the compound is classified as moderatelyactiveIf the IC₅₀ is <0.5 μg/ml, the compound is classified as active and isfurther evaluated using two strains, K1 and NF54. A new range ofconcentrations is chosen depending on the IC₅₀ determined (e.g. 100 to1.56 ng/ml) and the assay is carried out 2× independently.The standard drugs are chloroquine and artemisinin which are run in thesame assay. The IC₅₀ values for chloroquine are 2.9 ng/ml for NF54 and48 ng/ml for K1; for artemisinin 1.9 ng/ml for NF54 and 0.8 ng/ml forK1.

Secondary Screen

Test compounds are tested against a panel of say, 14 different ofdifferent origin and some show resistances to chloroquine and/orpyrimethamine. If the range of the IC₅₀ values for the 14 strains iswithin a factor 3-5× then the tested compound is considered not to showcross resistance.

Example B2 Malaria Enzyme Assays

Inhibition of Plasmodium falciparum dUTPase

Chemicals

2′-dUTP, was purchased from Pharmacia. MgCl₂, BSA, and the pH indicatorcresol red were from Sigma. The buffer N,N-bis(2-hydroxyethyl)glycine(BICINE) was obtained from USB (United States Biochemical), Ohio. Allthe concentrations of nucleotides were calculated spectrophotometrically(HP-8453, Hewlett Packard) at 280 nm, using the extinction coefficient(ε_(280 nm)=1.75 ml mg⁻¹ cm⁻¹). Other chemicals used in theseexperiments were of the highest quality available.

Cloning of the PFdut Gene

Conserved motifs of the human dUTPase enzyme were used as query toidentify the PFdut gene in the www.tigr.org database of the Plasmodiumfalciparum 3D7 strain. The entire coding sequence was amplified by thePCR using as template cDNA and as primers the oligonucleotides ATG-PFdut(CATATGCATTTAAAAATTGTATGTCTG) and TGA-PFdut(GGATCCTCAATATTTATTATCGATGTCGATC) which were designed so that NdeI andBamHI restriction sites were introduced at the 5′ and 3′ ends forconvenient cloning in the expression vector pET11 (Stratagene). Theamplified product was cloned into pGEMT (Promega) and propagated in E.coli XL1B cells. In order to confirm the correct sequence afteramplification, sequencing was performed using an Applied BiosystemsAutomated Sequencer, at the Analytical Services of the Instituto deParasitología y Biomedicina “López Neyra”. These Services also suppliedthe oligonucleotides designed for the sequencing

P. falciparum dUTPase Overexpression and Purification

Recombinant P. falciparum dUTPase was purified from E. coli BL21 (DE3)cells transformed with pET-PFdut. Pellets from a liter of culture wereresuspended in a solution consisting of buffer A (20 mM MES pH 5.5, 50mM NaCl, 1 mM DTT) plus the protease inhibitors 1 mM PMSF, 20 μg/mlleupeptin and 1 mM benzamidine. Purification was carried out in a coldroom (4° C.). The soluble crude extract was obtained by sonication in aVibra-cell (Sonics and Materials Inc. Danbury, Conn., USA) andcentrifugation at 14000×g. The extract was loaded onto aphosphocellulose column (Whatman) pre-equilibrated with buffer A at aflow rate of 1 ml/min. After washing the column with 100 ml of buffer A,elution was performed using a linear NaCl gradient of 50 to 1000 mM.Peak fractions with a low concentration of contaminating protein, asjudged by 15% SDS-PAGE gels, were pooled and then loaded andchromatographed on a Superdex 200 column at a flow rate of 0.5 ml/min.The column was equilibrated with buffer B (50 mM Bicine, 1 mM DTT, 10 mMMgCl₂). Peak fractions were pooled and concentrated to about 5 mg/ml byultrafiltration in a Centripep tube (Amicon) and stored at −80° C.

Kinetic Measurements

Nucleotide hydrolysis was monitored by mixing enzyme and substrate witha rapid kinetic accessory (Hi-Tech Scientific) attached to aspectrophotometer (Cary 50) and connected to a computer for dataacquisition and storage. Protons, released through the hydrolysis ofnucleotides, were neutralised by a pH indicator in a weak bufferedmedium with similar pK_(a) and monitored spectrophotometrically at theabsorbance peak of the basic form of the indicator. Absorbance changeswere kept within 0.1 units. The indicator/buffer pair used was cresolred/BICINE (2 mM/50 μM, pH 8, 573 nm). The measurements were performedat 25° C., and the solutions were previously degassed. Assays contained30 nM purified recombinant enzyme, 50 μM dUTP, 5 mM MgCl₂ and 2.5 mMDTT, 1.25 mg/ml BSA and 100 mM KCl. Indicator absorbance changescorresponding to complete hydrolysis of nucleotides were recorded in thecomputer, and the kinetic parameters V_(max) and K_(mapp) (or slope)were calculated by fitting the data to the integrated Michaelis-Mentenequation (Segel, 1975).

[dUMP]/t=V _(max) −K _(map) /t In [dUTP]/([dUTP]−[dUMP])

Solutions of potential inhibitors were prepared at 10 mg/ml and testedroutinely at concentrations of 2, 10, and 50 μg/μl. A wider range ofconcentrations was further tested when necessary for K_(i)determination. The different apparent K_(m) values attained were plottedagainst inhibitor concentration and K_(i) values were obtained accordingto the following equation:

$K_{map} = {{\frac{K_{m}}{K_{i}}\lbrack I\rbrack} + K_{m}}$

Example B3 Human dUTPase Assay

Human recombinant dUTPase was purified from E. coli BL21 (DE3) cellstransformed with pETHudut (Dr. P. O. Nyman, Lund University).Purification was accomplished as described for the dUTPase above exceptthat the last step in Superdex 200 was omitted. Likewise, conditions forenzyme assays were the same as described above except that the enzymeconcentration was 50 nM.

Example B4 Trypanosoma brucei Whole Cell Assays Parasite Cultures

Three strains of T. brucei spp. are used in this study: (a) Trypanosomabrucei rhodesiense STIB 900, a clone of a population isolated in 1982from a patient in Tanzania which is known to be susceptible to allcurrently used drugs; (b) Trypanosoma brucei gambiense STIB 930, aderivative of strain TH1/78E (031) isolated in 1978 from a patient inIvory Coast which is known to be sensitive to all drugs used, and (c)Trypanosoma brucei brucei STIB 950, a clone of a population isolated in1985 from a bovine in Somalia which shows drug resistance to diminazene,isometamidium and quinapyramine.The bloodstream form trypomastigotes of the strains a and c aremaintained in MEM medium with Earle's salts supplemented with 25 mMHEPES, 1 g/l additional glucose, 1% MEM non-essential amino acids(100×), 0.2 mM 2-mercaptoethanol, 2 mM Na-pyruvate, 0.1 mM hypoxanthineand 15% heat inactivated horse serum.The bloodstream form trypomastigotes of strain b are maintained in MEMmedium with Earle's salts supplemented with 25 mM HEPES, 1 g/ladditional glucose, 1% MEM non-essential aminoacids (100×), 0.2 mM2-mercaptoethanol, 2 mM Na-pyruvate, 0.1 mM hypoxanthine, 0.05 mMbathocuproine disulphonic acid, 0.15 mM L-cysteine and 15% heatinactivated pooled human serum.All cultures and assays are conducted at 37° C. under an atmosphere of5% CO₂ in air.

Drug Sensitivity Assays

Stock drug solutions are prepared in 100% DMSO (unless otherwisesuggested by the supplier) at 10 mg/ml, and heated or sonicated ifnecessary. After use the stocks are kept at −20° C. For the assays, thecompound is further diluted to the appropriate concentration usingcomplete medium.Assays are performed in 96-well microtiter plates, each well containing100 μl of culture medium with 8×10³ bloodstream forms with or without aserial drug dilution. The highest concentration for the test compoundsis 90 μg/ml. Seven 3-fold dilutions are used covering a range from 90μg/ml to 0.123 μg/ml. Each drug is tested in duplicate and each assay isrepeated at least once. After 72 hrs of incubation the plates areinspected under an inverted microscope to assure growth of the controlsand sterile conditions.10 μl of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilledwater) are now added to each well and the plates incubated for another 2hours. Then the plates are read with a Spectramax Gemini XS microplatefluorometer (Molecular Devices Cooperation, Sunnyvale, Calif., USA)using an excitation wave length of 536 nm and an emission wave length of588 nm. Data are analysed using the microplate reader software SoftmaxPro (Molecular Devices Cooperation, Sunnyvale, Calif., USA).

Primary Screen

The preliminary screen uses the Trypanosoma b. rhodesiense strain. Thecompounds are tested at 7 concentrations (drug concentrations rangingfrom 90 μg/ml to 0.123 μg/ml in 3-fold dilutions).If the IC₅₀ is >3 μg/ml, the compound is classified as inactiveIf the IC₅₀ is 0.2-3 μg/ml, the compound is classified as moderatelyactiveIf the IC₅₀ is <0.2 μg/ml, the compound is classified as activeThe standard drug is melarsoprol which is run in the same assay; theIC₅₀ for melarsoprol is 1.6 ng/ml.

Secondary Screen

Active compounds (IC₅₀<0.2 μg/ml) are tested against the Trypanosomabrucei gambiense STIB 930 and the drug resistant T. b. brucei STIB 950following the same protocol as described above.The standard drug is melarsoprol which is run in the same assay; theIC₅₀ for melarsoprol is 4.2 ng/ml for STIB 930 and 2.8 ng/ml for STIB950

Example B5 Trypanosoma cruzi Whole Cell Assays

Trypanosoma cruzi Cell Cultures:The Trypanosoma cruzi Tulahuen C2C4 strain, containing the

-galactosidase (Lac Z) gene, is used. The plasmid construct by Dr. S.Reed was obtained from Dr. F. Buckner, Seattle, as epimastigotes in LITmedium.The infective amastigote and trypomastigote stages are cultivated in L-6cells (rat skeletal myoblast cell line) in RPMI 1640 supplemented with 2mM L-glutamine and 10% heat-inactivated foetal bovine serum in 12.5 cm²tissue culture flasks. Amastigotes develop intracellularly,differentiate into trypomastigotes and leave the host cell. Thesetrypomastigotes infect new L-6 cells and are the stages used to initiatean infection in the assay. All cultures and assays are conducted at 37°C. under an atmosphere of 5% CO₂ in air.

Drug Sensitivity Assays

Stock drug solutions are prepared in 100% DMSO (dimethylsulfoxide)unless otherwise suggested by the supplier at 10 mg/ml, and heated orsonicated if necessary. The stocks are kept at −20° C. For the assays,the compound is further diluted to the appropriate concentration usingcomplete medium.Assays are performed in sterile 96-well microtiter plates, each wellcontaining 100 μl medium with 2×10³ L-6 cells. After 24 hours 50 μl of atrypanosome suspension containing 5×10³ trypomastigote bloodstream formsfrom culture are added to the wells. 48 hours later the medium isremoved from the wells and replaced by 100 μl fresh medium with orwithout a serial drug dilution. At this point the L-6 cells should beinfected with amastigotes and no free trypomastigotes should be in themedium. Seven 3-fold dilutions are used covering a range from 90 μg/mlto 0.123 μg/ml. Each drug is tested in duplicate. After 96 hours ofincubation the plates are inspected under an inverted microscope toassure growth of the controls and sterility.Then the substrate CPRG/Nonidet (50

l) is added to all wells. A colour reaction will become visible within2-6 hours and can be read photometrically at 540 nm. Data aretransferred into a graphic programme (e.g. EXCEL), sigmoidal inhibitioncurves determined and IC₅₀ values calculated.

Primary Screen

Benznidazole is used as the reference drug and shows an IC₅₀ value of0.34 μg/ml.If the IC₅₀ is >30 μg/ml, the compound is classified as inactive.If the IC₅₀ is between 2 and 30 μg/ml, the compound is classified asmoderately active.If the IC₅₀ is <2 μg/ml, the compound is classified as active.

Example B6 Leishmaniasis Macrophage In Vitro Screening Model Parasiteand Cell Cultures

The Leishmania.donovani strain MHOM/ET/67/L82 obtained from Dr. S.Croft, London) is used. The strain is maintained in the Syrian Goldenhamster. Amastigotes are collected from the spleen of an infectedhamster Amastigotes are grown in axenic culture at 37° C. in SM medium(Cunningham I., J. Protozool. 24, 325-329, 1977) at pH 5.4 supplementedwith 10% heat-inactivated foetal bovine serum under an atmosphere of 5%CO₂ in air.Primary peritoneal macrophages from NMRI mice are collected 1 day aftera macrophage production stimulation with an i.p injection of 2 ml of a2% potato starch suspension (FLUKA, Switzerland) All cultures and assaysare done at 37° C. under an atmosphere of 5% CO₂ in air.

Drug Sensitivity Assays

Stock drug solutions are prepared in 100% DMSO (unless otherwisesuggested by the supplier) at 10 mg/ml, and heated or sonicated ifnecessary. After use the stocks are kept at −20° C. For the assays, thecompound is further diluted in serum-free culture medium and finally tothe appropriate concentration in complete medium.Assays are performed in sterile 16-well chamber slides (LabTek,Nalgene/Nunc Int.) To each well 100 μl of a murine macrophage suspension(4×10⁵/ml) in RPMI 1640 (containing bicarbonate and HEPES) supplementedwith 10% heat inactivated fetal bovine serum is added. After 24 hrs 100μl of a suspension containing amastigotes (1.2×10⁶/ml) is addedresulting in a 3:1 ratio of amastigotes/macrophages. The amastigotes areharvested from an axenic amastigote culture and suspended in RPMI/FBS.24 hrs later, the medium containing free amastigotes is removed, washed1× and replaced by fresh medium containing four 3-fold drug dilutions.In this way 4 compounds can be tested on one 16-well tissue cultureslide. Untreated wells serve as controls. Parasite growth in thepresence of the drug is compared to control wells. After 4 days ofincubation the culture medium is removed and the slides fixed withmethanol for 10 min followed by staining with a 10% Giemsa solution.Infected and non-infected macrophages are counted for the controlcultures and the ones exposed to the serial drug dilutions. Theinfection rates are determined. The results are expressed as % reductionin parasite burden compared to control wells, and the IC₅₀ calculated bylinear regression analysis.

Primary Screen

The compounds are tested in duplicate at 4 concentrations ranging from 9to 0.3 μg/m. If the 1050 is below 0.3 μg/ml then the range is changed to1 to 0.03 μg/ml. Miltefosine is used as the reference drug and shows anIC₅₀ value of 0.325 μg/ml (0.22-0.42 μg/ml; n=4)If the IC₅₀ is higher than 10 μg/ml, the compound is classified asinactive.If the IC₅₀ is between 2 and 10 μg/ml, the compound is classified asmoderately active.If the IC₅₀ is <2 μg/ml, the compound is classified as active and isfurther evaluated in a secondary screening.

Drug Sensitivity Assays

Stock drug solutions are prepared in 100% DMSO (dimethylsulfoxide)unless otherwise suggested by the supplier at 10 mg/ml, and heated orsonicated if necessary. The stocks are kept at −20° C. For the assays,the compound is further diluted to the appropriate concentration usingcomplete medium.Assays are performed in sterile 96-well microtiter plates, each wellcontaining 100 μl medium with 2×10³ L-6 cells. After 24 hours 50 μl of atrypanosome suspension containing 5×10³ trypomastigote bloodstream formsfrom culture are added to the wells. 48 hours later the medium isremoved from the wells and replaced by 100 μl fresh medium with orwithout a serial drug dilution. At this point the L-6 cells should beinfected with amastigotes and no free trypomastigotes should be in themedium. Seven 3-fold dilutions are used covering a range from 90 μg/mlto 0.123 μg/ml. Each drug is tested in duplicate. After 96 hours ofincubation the plates are inspected under an inverted microscope toassure growth of the controls and sterility.Then the substrate CPRG/Nonidet (50 μl) is added to all wells. A colourreaction will become visible within 2-6 hours and can be readphotometrically at 540 nm. Data are transferred into a graphic programme(e.g. EXCEL), sigmoidal inhibition curves determined and IC₅₀ valuescalculated.

Primary Screen

Benznidazole is used as the reference drug and shows an IC₅₀ value of0.34 μg/ml.If the IC₅₀ is >30 μg/ml, the compound is classified as inactive.If the IC₅₀ is between 2 and 30 μg/ml, the compound is classified asmoderately active.If the IC₅₀ is <2 μg/ml, the compound is classified as active.

Example B7 Leishmania donovani, Axenic Amastigote Assay Parasite andCell Cultures:

The Leishmania donovani strain MHOM/ET/67/L82) is used. The strain ismaintained in the hamster. Amastigotes are collected from the spleen ofan infected hamster and adapted to axenic culture conditions at 37° C.The medium is a 1:1 mixture of SM medium (Cunningham I., J. Protozool.24, 325-329, 1977) and SDM-79 medium (Brun, R. & Schönenberger, M., ActaTrop. 36, 289-292, 1979) at pH 5.4 supplemented with 10%heat-inactivated FBS under an atmosphere of 5% CO₂ in air.

Drug Sensitivity Assays

Stock drug solutions are prepared in 100% DMSO (unless otherwisesuggested by the supplier) at 10 mg/ml, and heated or sonicated ifnecessary. After use the stocks are kept at −20° C. For the assays, thecompound is further diluted to the appropriate concentration usingcomplete medium.Assays are performed in 96-well microtiter plates, each well containing100 μl of culture medium with 10⁵ amastigotes from axecic culture withor without a serial drug dilution. The highest concentration for thetest compounds is 90 μg/ml. Seven 3-fold dilutions are used covering arange from 30 μg/ml to 0.041 μg/ml. Each drug is tested in duplicate andeach assay is repeated at least once. After 72 hours of incubation theplates are inspected under an inverted microscope to assure growth ofthe controls and sterile conditions.10 μl of Alamar Blue (12.5 mg resazurin dissolved in 1 L distilledwater) are now added to each well and the plates incubated for another 2hours. Then the plates are read with a Spectramax Gemini XS microplatefluorometer (Molecular Devices Cooperation, Sunnyvale, Calif., USA)using an excitation wave length of 536 nm and an emission wave length of588 nm.Data are analysed using the microplate reader software Softmax Pro(Molecular Devices Cooperation, Sunnyvale, Calif., USA).

Primary Screen

The compounds are tested in duplicate at 7 concentrations. Miltefosineis used as the reference drug and shows an IC₅₀ value of 0.12 μg/ml.If the IC₅₀ is >3 μg/ml, the compound is classified as inactiveIf the IC₅₀ is 0.1-3 μg/ml, the compound is classified as moderatelyactiveIf the IC₅₀ is <0.1 μg/ml, the compound is classified as active

Secondary Screen

Active and moderately active compounds are tested in the macrophageassay with intracellular amastigotes in their host cells, murinemacrophages.

Example B8 Biological Results

Compounds of the invention, such as those in the examples abovetypically show activities in the low micromolar range for Plasmodiumfalciparum enzyme (K_(i)) and cell culture (ED₅₀), with selectivity (SI)over the human enzyme of at least 10-fold:

R Ki uM SI ED₅₀ uM

Ph₃CO TBDPSO TPSO Ph₃CNH 1.8 4.2 2.8 0.2  10 191 324 230 6   6.6 1.1 4.5

Ph₃CO Ph₃NH 515    313    nd nd 1   1.8

TBDPSO TPSO 1.2 1.3 >833   >769   3.0 1.0

TBDPSO TPSO Ph₃CO Ph₃NH 89   975    5   12    9 nd  91 >83  8.8 1.0 2.05.3

ABBREVIATIONS

TBDPSO tert-butyldiphenylsilyloxy DMF dimethylformamide TPSOtriphenylsilyloxy DCM dichloromethane TBDMS tert-butyldimethylsilyl RTroom temperature THF tetrahydrofuran Ac acetyl TEA triethylamine LAHlithium- TLC thin layer chromatography aluminiumhydride DMAPdimethylaminopyridineThroughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

1. Use of a compound of formula I′, in the manufacture of a medicamentfor the treatment or prophylaxis of plasmodium infections in mammals,including man.

where A is O, S or CH₂; B is O, S or CHR³; R¹ is H, C₁-C₅ alkyl, C₂-C₅alkenyl, C₂-C₅ alkynyl or a 5 or 6 membered, saturated or unsaturatedring containing 0 to 3 heteroatoms selected from N, O and S, the alkyl,alkenyl, alkynyl or ring being independently optionally substituted withR⁴; R² is H, F; R³ is H, F, OH, NH₂ or a pharmaceutically acceptableester, amide or ether thereof; or R² and R³ together form a chemicalbond; D is —NHCO—, —CONH—, —O—, —C(═O)—, —CH═CH, —C═C—, —NR⁵—; R⁴ isindependently selected from hydrogen, halo, cyano, amino, nitro,carboxy, carbamoyl, hydroxy, oxo, C₁-C₅ alkyl, C₁-C₅ haloalkyl, C₁-C₅alkyloxy, C₁-C₅ alkanoyl, C₁-C₅ alkanoyloxy, C₁-C₅ alkylthio,—N(C₀-C₃-alkyl)₂, hydroxymethyl, aminomethyl, carboxymethyl;—SO_(n)N(C₀-C₃-alkyl), —SO_(n)C₁-C₅-alkyl, where n is 1 or 2; R⁵ is H,C₁-C₄ alkyl, C₁-C₄ alkanoyl; E is Si or C; R⁶, R⁷ and R⁸ areindependently selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,or a stable monocyclic, bicyclic or tricyclic ring system which issaturated or unsaturated in which each ring has 0 to 3 heteroatomsselected from N, O and S; R⁶, R⁷ and R⁸ are independently optionallysubstituted with R⁴; with the proviso that if R³ is H, OH, F, NH₂ or abond, then at least one of R⁶, R⁷ and/or R⁸ comprises an unsaturatedring; or a pharmaceutically acceptable salts thereof.
 2. Use accordingto claim 1, wherein A is —O— and B is —CHR³—, or A is —O— and B is —S—.3. Use according to claim 1, wherein R² and R³ form a chemical bond. 4.Use according to claim 1, wherein R³ is OH, NH₂ or F.
 5. Use accordingto claim 1, wherein R¹ is H.
 6. Use according to claim 1, whereinC₀-C₃-alkylene-D-C₀-C₃-alkylene is oxymethylene, oxyethylene oroxypropylene.
 7. Use according to claim 1, whereinC₀-C₃-alkylene-D-C₀-C₃-alkylene is aminomethylene, aminoethylene oraminopropylene.
 8. Use according to claim 1, wherein at least two of R⁶,R⁷ and R⁸ have an aromatic nature.
 9. Use according to claim 1, whereinR⁶ is optionally substituted phenyl.
 10. Use according to claim 9,wherein R⁸ is optionally substituted phenyl or pyridyl.
 11. Useaccording to claim 1, wherein E is C.
 12. A compound of the formula I

where A is O, S or CH₂; B is O, S or CHR³; R¹ is H, C₁-C₅ alkyl, C₂-C₅alkenyl, C₂-C₅ alkynyl or a 5 or 6 membered, saturated or unsaturatedring containing 0 to 3 heteroatoms selected from N, O and S, the alkyl,alkenyl, alkynyl or ring being independently optionally substituted withR⁴; R² is H, F; R³ is H, F, OH, NH₂ or a pharmaceutically acceptableester, amide or ether thereof; or R² and R³ together form a chemicalbond; D is ONHCO—, —CONH—, —O—, —C(═O)—, —CH═CH, —C═C—, —NR⁵—; R⁴ isindependently selected from hydrogen, halo, cyano, amino, nitro,carboxy, carbamoyl, hydroxy, oxo, C₁-C₅ alkyl, C₁-C₅ haloalkyl, C₁-C₅alkyloxy, C₁-C₅ alkanoyl, C₁-C₅ alkanoyloxy, C₁-C₅ alkylthio,—N(C₀-C₃-alkyl)₂, hydroxymethyl, aminomethyl, carboxymethyl;—SO_(n)N(C₀-C₃-alkyl), —SO_(n)C₁-C₅-alkyl, where n is 1 or 2; R⁵ is H,C₁-C₄-alkyl, C₁-C₄-alkanoyl; E is Si or C; R⁶ and R⁷ are independently astable monocyclic, bicyclic or tricyclic ring system which has anaromatic nature and wherein each ring has 0 to 3 heteroatoms selectedfrom N, O and S; R⁸ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or astable monocyclic, bicyclic or tricyclic ring system which is saturatedor unsaturated and in which each ring has 0 to 3 heteroatoms selectedfrom N, O and S; R⁶, R⁷ and R⁸ are independently optionally substitutedwith R⁴; with the proviso that if the group C₀-C₃alkyl-D-C₀-C₃ alkyl is—O—CH₂—, then the group E(R6)(R7)(R8) is not CPh₃ (trityl), methoxylatedtrityl or tert.butyldiphenylsilyl; and pharmaceutically acceptable saltsthereof.
 13. A compound according to claim 12, wherein A is —O— and B is—CHR³—, or A is —O and B is —S—.
 14. A compound according to claim 12,wherein R² and R³ form a chemical bond.
 15. A compound according toclaim 12, wherein R³ is OH, NH₂ or F.
 16. A compound according to claim12, wherein R¹ is H.
 17. A compound according to claim 12, whereinC₀-C₃-alkylene-D-C₀-C₃-alkylene is oxymethylene, oxyethylene oroxypropylene.
 18. A compound according to claim 12, whereinC₀-C₃-alkylene-D-C₀-C₃-alkylene is aminomethylene, aminoethylene oraminopropylene.
 19. A compound according to claim 12, wherein R⁶ isoptionally substituted phenyl.
 20. A compound according to claim 19wherein R⁷ is optionally substituted phenyl or pyridyl.
 21. A compoundaccording to claim 12 wherein E is C.
 22. A pharmaceutical compositioncomprising a compound as defined in claim 12 and a pharmaceuticallyacceptable carrier or diluent therefor.
 23. Use of a compound as definedin claim 12 in the manufacture of a medicament for the treatment orprophylaxis of parasite infections in mammals, including man.
 24. Useaccording to claim 23, wherein the parasite is a trypanosome orLeishmania species.